Methods and compositions for the treatment of radiation-related disorders

ABSTRACT

This invention relates, in part, to methods and compositions that are useful for the treatment and/or prevention of various disorders, including radiation-related disorders, such as acute radiation syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/197,408 (now U.S. Pat. No. 10,857,200), filedNov. 21, 2018, which is a continuation application of U.S. patentapplication Ser. No. 15/519,462 (now U.S. Pat. No. 10,183,056), filedApr. 14, 2017, which claims the benefit of U.S. Provisional PatentApplication Nos. 62/064,872, filed Oct. 16, 2014 and 62/214,572, filedSep. 4, 2015, the entire contents of which are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates, inter alia, to methods and compositions that areuseful for the treatment or prevention of various disorders, includingradiation-related disorders, such as acute radiation syndrome (ARS).

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:CLE014PC-Sequencelisting.txt; date recorded: Oct. 14, 2015; file size:4.61 KB).

BACKGROUND

With an increasing risk of nuclear and radiological emergencies, thereis a critical need for development of medical radiation countermeasures(MRC) which are safe, easily administered and effective in preventingand/or mitigating the potentially lethal tissue damage caused byradiation exposure, especially high-dose radiation exposure (e.g. ARS).There are currently no FDA approved MRCs. Commercially availablegranulocyte colony-stimulating factor (G-CSF, filgrastim) has been ofinterest as a MRC; however, G-CSF has only shown variably positiveresults in animal studies, has limited utility in mass-casualtysituations due to the need for multiple injections, supportive care, andlaboratory monitoring, and is not approved by FDA for this purpose. Theinflammatory cytokine interleukin (IL)-12 has also been evaluated, butdid not demonstrate consistently significant radiomitigation in animalstudies, has substantial inflammatory toxicity in humans, has notundergone a defined animal-to-human dose-conversion plan, and isunapproved by FDA for any use.

CBLB502 is a truncated derivative of the Salmonella flagellin proteinthat acts by triggering Toll-like receptor 5 (TLR5) signaling and hasshown great promise in development as a MRC. The efficacy of MRCs,including CBLB502, for preventing and/or mitigating the potentiallylethal tissue damage caused by radiation exposure is difficult to assesssince they cannot be ethically tested in humans. There remains a needfor safe and effective doses of MRCs, like CBLB502.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides for methods and compositionsthat are useful in preventing and/or mitigating the potentially lethaltissue damage caused by radiation exposure, including doses, regimens,and kits that comprise CBLB502 a/k/a/entolimod and provide safe andeffective treatment and/or prevention of ARS.

In one aspect, the present invention provides a method of treating orpreventing ARS, comprising administering an effective amount of CBLB502to a human patient, where the effective amount of CBLB502 is about 0.35to about 0.75 μg/kg or about 0.40 to about 0.60 μg/kg. In someembodiments, the effective amount of CBLB502 is about 0.4 to about 0.6μg/kg (e.g. about 0.4, or about 0.45, or about 0.5, or about 0.55, orabout 0.6 μg/kg). In some embodiments the human patient has been exposedor is at risk of being exposed to a high dose of radiation. In variousembodiments, the treatment reduces morbidity or mortality of an exposedpopulation of human patients or accelerates recovery from symptoms ofARS. In various embodiments, the human patient is administered CBLB502within one or more of the triage, emergency care, and definitive carestages of radiation and combined injuries, for example CBLB502 may beadministered within about 1 to about 48 hours, about 1 to about 25hours, or about 5 to about 20 hours, or about 10 to about 15 hours ofbeing exposed to radiation (e.g. within about 48 hours of being exposedto radiation, or within about 25 hours of being exposed to radiation,i.e. 48 hours or less or 25 hours or less after being exposed toradiation). In various embodiments the dose of CBLB502 is about 0.35μg/kg, or about 0.4 μg/kg, or about 0.45 μg/kg, or about 0.5 μg/kg, orabout 0.55 μg/kg, or about 0.6 μg/kg and may be slightly altered by thehuman patient's body weight (e.g. an absolute dose of CBLB502 of about 2μg for a pediatric human patient of about 0 to about 5 kg (e.g. about 0,or about 1, or about 2, or about 3, or about 4, or about 5 kg); or about3 μg for a pediatric human patient of about 6 to about 8 kg (e.g. about6, or about 7, or about 8 kg), or about 5 μg for a pediatric humanpatient of about 9 to about 13 kg (e.g. 9, or about 10, or about 11, orabout 12, or about 13 kg); or about 8 μg for a pediatric human patientof about 14 to about 20 kg (e.g. about 14, or about 16, or about 18, orabout 20 kg), or about 12 μg for a pediatric human patient of about 21to about 30 kg (e.g. about 21, or about 23, or about 25, or about 27, orabout 30 kg), or about 13 μg for a pediatric human patient of about 31to about 33 kg (e.g. about 31, or about 32, or about 33 kg), or about 20μg for an adult human patient of about 34 to about 50 kg (e.g. about 34,or about 36, or about 38, or about 40, or about 42, or about 44, orabout 46, or about 48, or about 50 kg), or about 30 μg for an adulthuman patient of about 51 to about 75 kg (e.g. about 51, or about 55, orabout 60, or about 65, or about 70, or about 75 kg), or about 45 μg foran adult human patient of greater than about 114 kg (e.g. about 114, orabout 120, or about 130, or about 140, or about 150 kg) In variousembodiments, CBLB502 is administered by injection (e.g. intramuscularinjection, or a single intramuscular injection).

In some embodiments, ARS comprises one of more of gastrointestinalsyndrome; hematopoietic syndrome; neurovascular syndrome;apoptosis-mediated tissue damage, wherein the apoptosis is optionallyattributable to cellular stress; and ionizing radiation inducedapoptosis tissue damage. In some embodiments, the high dose of radiation(e.g. ionizing radiation) is about 5 to about 30 Gy, or about 10 toabout 25 Gy, or about 15 to about 20 Gy and, optionally, sufficient fora classification of Unit Radiation Exposure Status of RES 3. In variousembodiments, the high dose of radiation is the result of a radiationdisaster and/or the human patient being treated has been exposed or isat risk of being exposed to a high dose of radiation as a result of oneor more of a military operation or a first responder operation in acontaminated area; a nuclear explosion; a criticality accident; aradiotherapy accident; a terrorist attack; exposure from space travel;escape of radioactive waste; exposure to open source radiation; and anuclear reactor malfunction.

In another aspect, the present invention provides a kit which issuitable for use upon exposure to a high dose of radiation, comprisingCBLB502, optionally formulated for intramuscular injection, in one ormore unit dosage forms of about 17.5 to about 47.5 μg (e.g. about 35 μg)and an injection needle. In some embodiments, the kit further comprisesinstructions for use and/or one or more of a radioactivity detector,potassium iodide (KI) or potassium iodate (KlO₃), gloves, face mask,hood, and cleaning solutions, and cleaning wipes. In variousembodiments, the kit is suitable for military field operations. In someembodiments, the present kits further comprise ibuprofen and/or a bottleof water for oral hydration.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sequence of CBLB502 (SEQ ID NO: 1).

FIG. 2 shows a 60-day survival dose-dependent study.

FIG. 3 shows improved survival of non-human primates (NHPs) injectedwith entolimod 1-48 hours after lethal irradiation. Kaplan-Meier plotsof non-human primate (NHP) survival over the 40 days following exposureto LD_(50/40)-LD_(75/40) doses of total body irradiation (TBI) areshown. Time frame of entolimod efficacy (panels A, B) was evaluated instudies Rs-03 (treatment at 1 hour after LD_(75/40) TBI; N=10) and Rs-06(treatment at 16, 25, or 48 hours after LD_(75/40) TBI; N=8-12).Dose-dependence of entolimod efficacy (panels C, D) was tested instudies Rs-09 (treatment at 1 hour after LD_(50/40)TBI; N=18) and Rs-14(treatment at 25 hours after LD_(50/40)TBI; N=10).

FIG. 4 shows accelerated hematological recovery of peripheral blood inNHPs injected with entolimod 16-48 hours post-irradiation. NHPs weretreated with a single injection of entolimod at 16-48 hours afterLD_(50/40) or LD_(75/40) TBI. Panels A, C, E, G: Effect of 40 μg/kgentolimod administered at different time points (16, 25 or 48 hours)after 6.5 Gy TBI (LD_(75/40); study Rs-06; N=8-12). Panels B, D, F, H:Effect of different entolimod doses (10 or 40 μg/kg) administered at 25hours after 6.75 Gy TBI (LD_(75/40); study Rs-14; N=10).Cytopenia/anemia thresholds: dotted lines—Grade 3 (platelets <50,000/μL;neutrophils <1,000/μL; hemoglobin <80 g/L); dashed lines—Grade 4(platelets <10,000/μL; neutrophils <500/μL; hemoglobin <65 g/L). Errorbars represent standard errors.

FIG. 5 shows accelerated recovery of the peripheral blood cellularityand hemoglobin content in NHPs irradiated with LD_(50/40) or LD_(75/40)of TBI and treated with different doses of entolimod 1 hour later.Panels A, C, E, G: study Rs-03; N=10. Panels B, D, F, H: study Rs-09;N=18. Cytopenia/anemia thresholds: dotted lines—Grade 3 (platelets<50,000/μL; neutrophils <1,000/μL; hemoglobin <80 g/L); dashedlines—Grade 4 (platelets <10,000/μL; neutrophils <500/μL; hemoglobin <65g/L). Error bars represent standard errors.

FIG. 6 shows enhanced morphological recovery of hematopoietic andlymphoid organs in NHPs treated with entolimod post-irradiation. NHPswere treated with a single injection of 40 μg/kg entolimod 16, 25 or 48hours after LD_(75/40) total body irradiation (TBI). Tissue morphologywas assessed 40 days post-irradiation and compared to that in controlNHPs treated with vehicle 16 hours after LD_(75/40) TBI. Representativehistological images (hematoxylin-eosin staining) of sternum bone marrowsections, thymuses, spleens and mesenteric lymph nodes of animals thatsurvived to study termination on Day 40 post-TBI (study Rs-06) areshown. Scale bars: 100 μm for bone marrow, 200 μm for thymus, spleen,and lymph node.

FIG. 7 shows comparable restorative effects of single 10 or 40 μg/kgentolimod treatments given 25 hours after TBI on morphological recoveryof hematopoietic and lymphoid organs in NHPs 40 days after irradiationwith LD_(50/40) of TBI. Representative histological images(hematoxylin-eosin staining) of sternum bone marrow sections, thymuses,spleens and mesenteric lymph nodes from animals that survived to studytermination on Day 40 post-TBI (study Rs-14). Scale bars: 100 μm forbone marrow, 200 μm for thymus, spleen, and lymph node.

FIG. 8 shows accelerated kinetics of bone marrow regeneration andproliferating phenotype of CFU-GM colonies in entolimod-treated CD2F1mice after LD_(50/30) of TBI and entolimod treatment. Male CD2F1 micewere irradiated with 9 Gy TBI and injected i.m. with vehicle or 200μg/kg entolimod 25 h later. Groups of 5 mice were sacrificed at theindicated time points for evaluation of histopathology and bone marrowclonogenic potential. Panel A: Representative microphotographs ofhematoxylin-eosin-stained bone marrow sections. Scale bar—100 μm. PanelB: Appearance of CFU-GM colonies grown from bone marrow collected on day7 after TBI.

FIG. 9 shows entolimod treatment ameliorates radiation damage in thegastrointestinal (GI) tract. Panels A, B: Small intestine sections fromNHPs 8 hours after exposure to 6.5 Gy TBI and treatment with vehicle or40 μg/kg entolimod 1 h later (study Rs-04). Blue—DAPI nuclear staining,red—smooth muscle actin immunostaining. Panel A: TUNEL staining showingfewer apoptotic cells (green) in GI crypts of entolimod-treated NHPs(scale bar 100 μm); Panel B: SOD2 immunostaining (green) showing morepositive cells in GI villi (arrowheads) and lamina propria (arrows) ofentolimod-treated NHPs (scale bar 50 μm). Panels C, D: Small intestinesections of NHPs 7 days after exposure to 11 Gy TBI and treatment withvehicle or 40 μg/kg entolimod 4 h later (study Rs-22). Panel C:Visualization of proliferating cells in the jejunum crypts: EdU (10mg/kg i.v. 1 h before euthanasia) inclusion in replicating DNA (green)and phosphohistone 3 immunostaining of mitotic cells (red) showing moreintensive proliferation of GI crypts in entolimod-treated NHPs (scalebar—200 μm). Panel D: H&E staining of ileum sections: upper panels—lowmagnification (scale bar—200 μm), lower panels—high magnification (scalebar—50 μm).

FIG. 10 shows improved GI tract morphology in NHPs irradiated with 6.5Gy TBI and treated with a single injection of entolimod at 1, 16, or 25hours later. Rhesus macaques were injected i.m. with vehicle or 40 μg/kgentolimod 1, 16 or 25 h after 6.5 Gy TBI (study Rs-08). Samples werecollected on day 5 after TBI for H&E staining. Scale bars—200 μm fororal mucosa and oropharynx; 100 μm—for all other GI tract segments.

FIG. 11 shows improved preservation of intestinal innervation andmuscularis mucosae integrity in the GI tract of irradiated NHPs treatedwith entolimod 1 hour after 6.5 Gy TBI. Rhesus macaques were injectedi.m. with vehicle or 40 μg/kg entolimod 1 h after 6.5 Gy TBI andduodenum samples were collected 5 days later (study Rs-08). Upperpanels: arrows point to disruptions of muscularis mucosae (red)—presentmostly in vehicle-treated NHPs. Bottom panels: arrows point to axons andneural termini (green) in the cryptal area of the small intestine—moreabundant in entolimod-treated NHPs.

FIG. 12 shows the effect of entolimod treatment on G-CSF and IL-6 levelsin peripheral blood of irradiated NHPs. Panels A, B: Effect of differententolimod doses administered 1 h after LD_(50/40) TBI (6.75 Gy; studyRs-09; N=18). Panels C, D: Effect of different entolimod dosesadministered 25 h after LD_(50/40) TBI (6.75 Gy; study Rs-14; N=10).Panels E, F: Comparison of dose-dependence of background-adjusted AreaUnder the Curve (AUC₀₋₂₄) values for G-CSF and IL-6 after entolimodtreatment given 1 h versus 25 h after LD_(50/40) TBI (with dashedlog-linear regression lines). Error bars represent standard errors.

FIG. 13 shows the effect of single dose entolimod treatment on IL-8 andIL-10 levels in the peripheral blood of NHPs irradiated with LD_(50/40)or LD_(75/40) doses of TBI. Panels A, B: Effects of different entolimoddoses administered 1 h after TBI (study Rs-09; N=18). Panels C, D:Effect of different entolimod doses administered 25 h after TBI (studyRs-14; N=10). Error bars represent standard errors.

FIG. 14 shows entolimod concentrations in the peripheral blood of NHPs(irradiated with LD_(50/40) (6.75 Gy) TBI or non-irradiated) atdifferent times after single intramuscular injection of the indicateddrug doses. Panel A: Entolimod levels after injection of different doses1 h after TBI (study Rs-09; N=18). Panel B: Entolimod levels afterinjection of the same dose levels in non-irradiated NHPs (study04-Rs-04; N=6). Error bars represent standard errors.

FIG. 15 is, without wishing to be bound by theory, a schematicpresentation of mechanism(s) underlying anti-acute radiation syndrome(ARS) effects of entolimod. Entolimod binding to Toll-like receptor 5(TLR5) initiates a cascade of events, all merging at attenuation ofmajor pathological processes—leading causes of death in ARS: damage tohematopoietic (HP) and gastrointestinal (GI) systems resulting inbleeding and sepsis. The immediate TLR5-dependent effectors includeanti-oxidants (e.g., SOD2), anti-apoptotic factors (both NF-κB-dependent(i.e., IAP and Bcl family members), and NF-κB-independent (i.e.,PI3K/AKT, MKP7 and STAT3), hematopoietic cytokines (e.g., G-CSF andIL-6), anti-infective factors and processes (e.g., neutrophilmobilization). In addition, stimulation of TLR5 is expected to inhibitradiation-induced aseptic inflammation involved in secondary tissuedamage e.g. via induction of an anti-inflammatory cytokine IL-10, IL-1βantagonist (IL-1βa) and stimulation of mesenchymal stem cells (MSC)known to express TLR5 and to have anti-inflammatory properties. Togetherwith fibroblasts that can be induced to proliferate via TLR5stimulation, MSC may also contribute to wound-healing processes. Dashedlines show all molecular connections downstream of TLR5 that are notdirectly established for entolimod, but are extrapolated from publisheddata on TLR5-dependent effects of flagellin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of effectiveand safe doses of CBLB502 for use in humans for the treatment of ARS.The present inventors have surprisingly discovered that the human doseis less than 1 μg/kg, which is far less than conventional biologics(which are often administered in the mg/kg range) and far less thanprior animal studies on CBLB502 (e.g. Science 320, 226 (2008) and JPharmacol Exp Ther. 343(2):497-508 (2012) (the contents of which arehereby incorporated by reference in their entireties).

In one aspect, the present invention provides a method of treating orpreventing ARS, comprising administering an effective amount of CBLB502to a human patient in need thereof, where the effective amount ofCBLB502 is about 0.35 to about 0.75 μg/kg. In some embodiments, theeffective amount of CBLB502 is about 0.4 to about 0.6 μg/kg (e.g. about0.4, or about 0.45, or about 0.5, or about 0.55, or about 0.6 μg/kg). Insome embodiments, the effective amount of CBLB502 is about 2 μg/subjectto about 45 μg/subject, or about 5 to about 40, or about 10 to about 30,or about 15 to about 25 μg/subject. In another aspect, the presentinvention provides for use of about 0.35 to about 0.75 μg/kg of CBLB502(e.g. about 0.4 to about 0.6 μg/kg) in the treatment or prevention ofARS. In another aspect, the invention provides a use of about 0.35 toabout 0.75 μg/kg of CBLB502 (e.g. about 0.4 to about 0.6 μg/kg) in themanufacture of a medicament for the treatment or prevention of ARS.

In one aspect, the present invention provides a method of reducing therisk of death following exposure to potentially lethal irradiation (forinstance, 2 Gy) occurring as the result of a radiation disastercomprising administering an effective amount of CBLB502 to a humanpatient in need thereof. In various embodiments, the effective amount ofCBLB502 is about 0.4 to about 0.6 μg/kg (e.g. about 0.4, or about 0.45,or about 0.5, or about 0.55, or about 0.6 μg/kg).

CBLB502 is a flagellin-related polypeptide (see, e.g., FIG. 7 of U.S.Patent Publication No. 2003/0044429, the contents of which areincorporated herein by reference in their entirety). As used herein“CBLB502” (aka “Entolimod”) refers to a polypeptide which comprises thesequence of SEQ ID NO: 1 or a sequence of about 95%, or about 96%, orabout 97% or about 98%, or about 99% sequence similarity thereto.

In some embodiments, CBLB502 activates TLR5 signaling and, optionally,activation of TLR5 induces expression of the nuclear factor NF-κB, whichin turn activates numerous targets, including inflammatory-relatedcytokines.

In further embodiments, CBLB502 induces expression of proinflammatorycytokines. In further embodiments, CBLB502 induces expression ofanti-inflammatory molecules. In another embodiment, CBLB502 inducesexpression of anti-apoptotic molecules. In yet a further embodiment,CBLB502 induces expression of anti-bacterial molecules. Targets ofNF-κB, include, but are not limited to, IL-β, TNF-α, IL-6, IL-8, IL-18,G-CSF, TNFSF13B, keratinocyte chemoattractant (KC), BLIMP1/PRDM1, CCL5,CCL15, CCL17, CCL19, CCL20, CCL22, CCL23, CXCL1, CCL28, CXCL11, CXCL10,CXCL3, CXCL1, GRO-beta, GRO-gamma, CXCL1, ICOS, IFNG, IL-1A, IL-1B,IL1RN, IL-2, IL-9, IL-10, IL-11, IL-12, IL-12B, IL-12A, IL-13, IL-15,IL-17, IL-23A, IL-27, EBI3, IFNB1, CXCL5, KC, IiGp1, CXCL5, CXCL6, LTA,LTB, CCL2, CXCL9, MCP-1/JE, CCL3, CCL4, CXCL3, CCL20, CXCL10, CXCL5,CCL5, CCL1, TNF beta, TNFSF10, TFF3, TNFSF15, CD86, complement component8a, CCL27, defensin-63, MIG, MIP-2, and/or NOD2/CARD15. Targets ofNF-κB, include, but are not limited to G-CSF and IL-6. In someembodiments, any of these targets find use as biomarkers (e.g. fordosing and/or determination of presence and/or extent of aradiation-based disorder). Further biomarkers include numbers, includingrelative numbers, of blood cell counts, including but not limited to,lymphocytes, neutrophils, platelets, and ratio of neutrophils tolymphocytes.

In some embodiments, CBLB502 is administered to a human patient at aneffective amount (a.k.a. dose) of less than about 1 μg/kg, for instance,about 0.35 to about 0.75 μg/kg or about 0.40 to about 0.60 μg/kg. Insome embodiments, the dose of CBLB502 is about 0.35 μg/kg, or about 0.40μg/kg, or about 0.45 μg/kg, or about 0.50 μg/kg, or about 0.55 μg/kg, orabout 0.60 μg/kg, or about 0.65 μg/kg, or about 0.70 μg/kg, or about0.75 μg/kg, or about 0.80 μg/kg, or about 0.85 μg/kg, or about 0.90μg/kg, or about 0.95 μg/kg or about 1 μg/kg. In various embodiments, theabsolute dose of CBLB502 is about 2 μg/subject to about 45 μg/subject,or about 5 to about 40, or about 10 to about 30, or about 15 to about 25μg/subject. In some embodiments, the absolute dose of CBLB502 is about20 μg, or about 30 μg, or about 40 μg.

In various embodiments, the dose of CBLB502 may be determined by thehuman patient's body weight. For example, an absolute dose of CBLB502 ofabout 2 μg for a pediatric human patient of about 0 to about 5 kg (e.g.about 0, or about 1, or about 2, or about 3, or about 4, or about 5 kg);or about 3 μg for a pediatric human patient of about 6 to about 8 kg(e.g. about 6, or about 7, or about 8 kg), or about 5 μg for a pediatrichuman patient of about 9 to about 13 kg (e.g. 9, or about 10, or about11, or about 12, or about 13 kg); or about 8 μg for a pediatric humanpatient of about 14 to about 20 kg (e.g. about 14, or about 16, or about18, or about 20 kg), or about 12 μg for a pediatric human patient ofabout 21 to about 30 kg (e.g. about 21, or about 23, or about 25, orabout 27, or about 30 kg), or about 13 μg for a pediatric human patientof about 31 to about 33 kg (e.g. about 31, or about 32, or about 33 kg),or about 20 μg for an adult human patient of about 34 to about 50 kg(e.g. about 34, or about 36, or about 38, or about 40, or about 42, orabout 44, or about 46, or about 48, or about 50 kg), or about 30 μg foran adult human patient of about 51 to about 75 kg (e.g. about 51, orabout 55, or about 60, or about 65, or about 70, or about 75 kg), orabout 45 μg for an adult human patient of greater than about 114 kg(e.g. about 114, or about 120, or about 130, or about 140, or about 150kg).

In various embodiments, CBLB502 is administered within about 1 to about48 hours, or about 1 to about 25 hours, or about 5 to about 20 hours, orabout 10 to about 15 hours of a human patient being exposed toradiation. In some embodiments, CBLB502 is administered within about 1,or about 2, or about 3, or about 4, or about 5, or about 6, or about 7,or about 8, or about 9, or about 10, or about 11, or about 12, or about13, or about 14, or about 15, or about 16, or about 17, or about 18, orabout 19, or about 20, or about 21, or about 22, or about 23, or about24, or about 25, or about 26, or about 27, or about 28, or about 29, orabout 30, or about 31, or about 32, or about 33, or about 34, or about35, or about 36, or about 37, or about 38, or about 39, or about 40, orabout 41, or about 42, or about 43, or about 44, or about 45, or about46, or about 47, or about 48 hours of a human patient being exposed toradiation. In one embodiment, a human patient is administered CBLB502within about 25 hours of being exposed to radiation. In anotherembodiment, a human patient is administered CBLB502 within about 48hours of being exposed to radiation.

The medical management of radiation and combined injuries can be dividedinto three stages: triage, emergency care, and definitive care. Duringtriage, patients are prioritized and rendered immediate lifesaving care.Emergency care includes therapeutics and diagnostics necessary duringthe first 12 to 24 hours. Definitive care is rendered when finaldisposition and therapeutic regimens are established. In one embodiment,CBLB502 is administered as part of the triage emergency care stages. Forexample, it may be used during a military field operation as describedherein and, optionally, may find use in a kit. In various embodiments,CBLB502 is administered within one or more of the triage, emergencycare, and definitive care stages of radiation and combined injuries.

Administration of CBLB502 (and/or additional agents) described hereincan, independently, be one to four times daily or one to four times permonth or one to six times per year or once every two, three, four orfive years.

Administration can be for the duration of one day or one month, twomonths, three months, six months, one year, two years, three years, andmay even be for the life of the human patient. The dosage may beadministered as a single dose or divided into multiple doses. In someembodiments, CBLB502 is administered about 1 to about 3 times (e.g. 1,or 2 or 3 times). In some embodiments, CBLB502 is administered once. Insome embodiments, CBLB502 (and/or additional agents) described herein isadministered as a slow IV infusion or drip (e.g. over about 0.5, orabout 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 5,or about 10 hours).

Various modes of administration of CBLB502 and additional agents aredisclosed herein. In one embodiment, CBLB502 is administeredparenterally. In some embodiments, CBLB502 is administered by injection,e.g. intramuscular injection. In some embodiments, CBLB502 is by asingle intramuscular injection. In some embodiments, administration isaccomplished using a kit as described herein (e.g. via a unit dose form,e.g. a pre-loaded (a.k.a. pre-dosed or pre-filled) syringe or a penneedle injector (injection pen)).

In various embodiments, the present methods and compositions providetreatment or prevention of radiation-related disorders, such as ARS. Invarious embodiments, the treatments described herein reduce morbidity ormortality of an exposed population of human patients or acceleratesrecovery from symptoms of ARS. ARS often presents as a sequence ofphased symptoms, which may vary with individual radiation sensitivity,type of radiation, and the radiation dose absorbed. Generally, withoutwishing to be bound by theory, the extent of symptoms will heighten andthe duration of each phase will shorten with increasing radiation dose.ARS can be divided into three phases: prodromal phase (a.k.a. N-V-Dstage), latent period and manifest illness. In various embodiments,CBLB502, as describe herein, may be administered to a human patient inany one of these three stages (i.e. CBLB502 may be administered to ahuman patient in the prodromal phase, CBLB502 may be administered to ahuman patient in latent period, or CBLB502 may be administered to ahuman patient in manifest illness stage).

In the prodromal phase there is often a relatively rapid onset ofnausea, vomiting, and malaise. Use of antiemetics, (e.g. oralprophylactic antiemetics) such as granisetron (KYTRIL), ondansetron(ZOFRAN), and 5-HT3 blockers with or without dexamethasone, may beindicated in situations where high-dose radiological exposure hasoccurred, is likely, or is unavoidable. Accordingly, in variousembodiments, CBLB502 may be administered to a human patient in receivingan anti-emetic agent or CBLB502 may be administered to a human patientin combination with an anti-emetic agent. For example, CBLB502 may alsobe added to the following antiemetic regimens: Ondansetron: initially0.15 mg/kg IV; a continuous IV dose option consists of 8 mg followed by1 mg/h for the next 24 hours. Oral dose is 8 mg every 8 hours as neededor Granisetron (oral dosage form): dose is usually 1 mg initially, thenrepeated 12 hours after the first dose. Alternatively, 2 mg may be takenas one dose. IV dose is based on body weight; typically 10 μg/kg (4.5μg/lb) of body weight.

In the latent period, a human patient may be relatively symptom free.The length of this phase varies with the dose. The latent phase islongest preceding the bone-marrow depression of the hematopoieticsyndrome and may vary between about 2 and 6 weeks. The latent period issomewhat shorter prior to the gastrointestinal syndrome, lasting from afew days to a week. It is shortest of all preceding the neurovascularsyndrome, lasting only a matter of hours. These times are variable andmay be modified by the presence of other disease or injury. Manifestillness presents with the clinical symptoms associated with the majororgan system injured (marrow, intestinal, neurovascular).

In some embodiments, the present invention relates to the mitigation of,or protection of cells from, the effects of exposure to radiation. Insome embodiments, the present invention pertains to a method ofmitigating and/or protecting a human patient from radiation comprisingadministering CBLB502 at the disclosed doses and regimens (and/oradditional agents) described herein. In some embodiments, the radiationis ionizing radiation. In some embodiments, the ionizing radiation issufficient to cause gastrointestinal syndrome or hematopoietic syndrome.

In some embodiments, the ARS comprises one of more of gastrointestinalsyndrome; hematopoietic syndrome; neurovascular syndrome;apoptosis-mediated tissue damage, wherein the apoptosis is optionallyattributable to cellular stress; and ionizing radiation inducedapoptosis tissue damage.

Hematopoietic syndrome (a.k.a. bone marrow syndrome) is characterized byloss of hematopoietic cells and their progenitors making it impossibleto regenerate blood and lymphoid system. This syndrome is often markedby a drop in the number of blood cells, i.e., aplastic anemia. This mayresult in infections (e.g. opportunistic infections) due to a low amountof white blood cells, bleeding due to a lack of platelets, and anemiadue to few red blood cells in the circulation. These changes can bedetected by blood tests after receiving a whole-body acute dose.Conventional trauma and burns resulting from a bomb blast arecomplicated by the poor wound healing caused by hematopoietic syndrome,increasing mortality. Death may occur as a consequence of infection(result of immunosuppression), hemorrhage and/or anemia. Hematopoieticsyndrome usually prevails at the lower doses of radiation and leads tothe more delayed death than GI syndrome.

Gastrointestinal syndrome is caused by massive cell death in theintestinal epithelium, predominantly in the small intestine, followed bydisintegration of intestinal wall and death from bacteriemia and sepsis.Symptoms of this form of radiation injury include nausea, vomiting, lossof appetite, loss of absorptive capacity, hemorrhage in denuded areas,and abdominal pain. Illustrative systemic effects of gastrointestinalsyndrome include malnutrition, dehydration, renal failure, anemia,sepsis, etc. Without treatment (including, for example, bone marrowtransplant), death is common (e.g. via infection from intestinalbacteria). In some embodiments, CBLB502, at the doses and regimensdescribed herein, may be used in combination with bone marrowtransplant. In some embodiments, CBLB502, at the doses and regimensdescribed herein, may be used in combination with one or more inhibitorsof GI syndrome and/or any of the additional agents described herein.

Neurovascular syndrome presents with neurological symptoms such asdizziness, headache, or decreased level of consciousness, occurringwithin minutes to a few hours, and with an absence of vomiting.Additional symptoms include extreme nervousness and confusion; severenausea, vomiting, and watery diarrhea; loss of consciousness; andburning sensations of the skin. Neurovascular syndrome is commonlyfatal.

In some embodiments, the present invention provides a method forreducing the risk of death following exposure to irradiation comprisingadministering an effective amount of CBLB502. In some embodiments, theradiation is potentially lethal, and, optionally, occurs as the resultof a radiation disaster. In various embodiments, CBLB502 is administeredwithin 25 hours following radiation exposure. In various embodiments,CBLB502 is administered within 48 hours following radiation exposure. Insome embodiments, the present invention provides a method for reducingthe risk of death following exposure to potentially lethal irradiationoccurring as the result of a radiation disaster, comprisingadministering CBLB502 within 25 hours following radiation exposure. Insome embodiments, the present invention provides a method for reducingthe risk of death following exposure to potentially lethal irradiationoccurring as the result of a radiation disaster, comprisingadministering CBLB502 within 48 hours following radiation exposure

In some embodiments, CBLB502 at the disclosed doses and regimens is usedto treat a disorder linked to apoptosis which is attributable tocellular stress (see, e.g. U.S. Pat. Nos. 7,638,485 and 8,106,005, thecontents of which are hereby incorporated by reference in theirentirety). In some embodiments, CBLB502 (and/or additional agents)described herein are administered prior to, together with, or after thetissue damage. In some embodiments, the cellular stress is radiation. Insome embodiments, CBLB502 (and/or additional agents) are administered incombination with any additional agent described herein, including butnot limited to a radioprotectant (e.g. an antioxidant (e.g. amifostineand vitamin E), a cytokine (e.g. a stem cell factor)), etc. Injury anddeath of normal cells from ionizing radiation is a combination of adirect radiation-induced damage to the exposed cells and an activegenetically programmed cell reaction to radiation-induced stressresulting in a suicidal death or apoptosis. Apoptosis plays a key rolein massive cell loss occurring in several radiosensitive organs (e.g.,hematopoietic and immune systems, epithelium of digestive tract, etc.),the failure of which determines general radiosensitivity of theorganism. In some embodiments, administration of CBLB502 (and/oradditional agents) of the invention to a human patient in need thereofsuppresses apoptosis in cells. In some embodiments, CBLB502 (and/oradditional agents) of the invention are administered to a human patientto protect healthy cells from the damaging effects of the radiationtreatment.

In various embodiments, the present invention provides a method forreducing apoptosis following exposure to irradiation. In an embodiment,the present invention provides a method for reducing apoptosis ofhematopoietic cells following irradiation. In another embodiment, thepresent invention provides a method for reducing apoptosis ofgastrointestinal cells following irradiation.

In various embodiments, administration of CBLB502 at the discloseddosages stimulates and protects stem cells. For example, the presentinvention and composition may stimulate and protect hematopoietic stemcells including various hematopoietic progenitor cells. In anotherexample, the present invention and composition may stimulate and protectgastrointestinal stem cells such as intestinal crypt stem cells. In someembodiments, the stem cells may be stimulated to proliferate andregenerate. Accordingly the present invention provides methods ofexpanding the number of stem cells such as hematopoietic stem cells orgastrointestinal stem cells in a patient. In some embodiments,hematopoietic progenitor cells or gastrointestinal progenitor cells areexpanded. In various embodiments, the present invention provides methodsand compositions that protect the stem cells or progenitors cells fromcell death (e.g., apoptosis or necrosis).

In various embodiments, methods and compositions of the presentinvention significantly enhances recovery of the hematopoietic and GIsystems following irradiation. For example, methods and compositions ofthe present invention enhances bone marrow recovery followingirradiation. In another example, methods and compositions of the presentinvention enhances regeneration of the GI crypt.

Exposure to ionizing radiation (IR) may be short- or long-term, and/orit may be experienced as a single or multiple doses and/or it may beapplied to the whole body or locally. The present invention, in someembodiments, pertains to nuclear accidents or military attacks, whichmay involve exposure to a single high dose of whole body irradiation(sometimes followed by a long-term poisoning with radioactive isotopes),as further described herein. The same is true (with strict control ofthe applied dose), for example, for pretreatment of patients for bonemarrow transplantation when it is necessary to prepare hematopoieticorgans for donor's bone marrow by “cleaning” them from the host bloodprecursors. Cancer treatment may involve multiple doses of localirradiation that greatly exceeds lethal dose if it were applied as atotal body irradiation (e.g. a radiotherapy accident). Poisoning ortreatment with radioactive isotopes results in a long-term localexposure to radiation of targeted organs (e.g., thyroid gland in thecase of inhalation of ¹²⁵I). Further, there are many physical forms ofionizing radiation differing significantly in the severity of biologicaleffects.

At the molecular and cellular level, radiation particles are able toproduce breakage and cross-linking in the DNA, proteins, cell membranesand other macromolecular structures. Ionizing radiation also induces thesecondary damage to the cellular components by giving rise to the freeradicals and reactive oxygen species (ROS). Multiple repair systemscounteract this damage, such as, several DNA repair pathways thatrestore the integrity and fidelity of the DNA, and antioxidant chemicalsand enzymes that scavenge the free radicals and ROS and reduce theoxidized proteins and lipids. Cellular checkpoint systems detect the DNAdefects and delay cell cycle progression until damage is repaired ordecision to commit cell to growth arrest or programmed cell death(apoptosis) is reached

Radiation can cause damage to mammalian organism ranging from mildmutagenic and carcinogenic effects of low doses to almost instantkilling by high doses. Overall radiosensitivity of the organism isdetermined by pathological alterations developed in several sensitivetissues that include hematopoietic system, reproductive system anddifferent epithelia with high rate of cell turnover.

Acute pathological outcome of gamma irradiation leading to death isdifferent for different doses and may be determined by the failure ofcertain organs that define the threshold of organism's sensitivity toeach particular dose. Thus, lethality at lower doses occurs from bonemarrow aplasia, while moderate doses kill faster by inducing agastrointestinal (GI) syndrome. Very high doses of radiation can causealmost instant death eliciting neuronal degeneration. Organisms thatsurvive a period of acute toxicity of radiation can suffer fromlong-term remote consequences that include radiation-inducedcarcinogenesis and fibrosis developing in exposed organs (e.g., kidney,liver or lungs) in the months and years after irradiation. Cellular DNAis a major target of IR that causes a variety of types of DNA damage(genotoxic stress) by direct and indirect (e.g. free radical-based)mechanisms. All organisms maintain DNA repair system capable ofeffective recovery of radiation-damaged DNA; errors in DNA repairprocess may lead to mutations.

CBLB502, at the doses and regimens described herein, possesses strongpro-survival activity at the cellular level and on the organism as awhole. In response to super-lethal doses of radiation, CBLB502 mayinhibit both gastrointestinal and hematopoietic syndromes, which aremajor causes of death from acute radiation exposure. As a result ofthese properties, CBLB502 may be used to treat the effects of naturalradiation events and nuclear accidents. Moreover, CBLB502 can be used incombination with other radioprotectants, thereby, dramaticallyincreasing the scale of protection from ionizing radiation.

CBLB502, at the doses and regimens described herein, may be used as aradioprotective agent to extend the range of tolerable radiation dosesby, for example, increasing radioresistance of human organism beyond thelevels achievable by currently available measures (shielding andapplication of existing bioprotective agents) and drastically increasethe chances of crew survival in case of nuclear accidents or large-scalesolar particle events, for example.

CBLB502, at the doses and regimens described herein, may inhibitradiation-induced programmed cell death or apoptosis in response todamage in DNA and other cellular structures. In some embodiments,CBLB502 as described herein may not deal with damage at the cellularlevel and may not prevent mutations. Free radicals and reactive oxygenspecies (ROS) are the major cause of mutations and other intracellulardamage. Antioxidants and free radical scavengers are effective atpreventing damage by free radicals.

Further, in some embodiments, the present invention relates to theprevention or treatment of cutaneous radiation syndrome (CRS), i.e. skinsymptoms of radiation exposure (e.g. redness (optionally associated withitching), blistering, ulceration, hair loss, damaged sebaceous and sweatglands, atrophy, fibrosis, decreased or increased skin pigmentation,ulceration or necrosis of the exposed tissue moist desquamation andcollapse of the dermal vascular system after two months, resulting inthe loss of the full thickness of the exposed skin.

In various embodiments, administration of CBLB502 at the discloseddosages reduces the incidence of wounds, septic complications, andmicrobial infections in patients following irradiation.

In some embodiments, the present human patients experience leukopeniaand/or neutropenia (e.g. absolute neutrophil count (ANC)<100 cells/μL.In some embodiments, the present methods and compositions pertain to ahuman patient which presents a lymphocyte count reduction of about 50%within about 24 to about 48 hours. In some embodiments, the humanpatient's lymphocyte count is less than about 1000 cells/μL, or about900 cells/μL, or about 800 cells/μL, or about 700 cells/μL, or about 600cells/μL, or about 500 cells/μL, or about 400 cells/μL, or about 300cells/μL, or about 200 cells/μL, or about 100/cells μL (e.g. withinabout 24 to about 48 hours). In some embodiments, the patient'slymphocyte profile is assessed by the Andrews Lymphocyte Nomogram (seeAndrews G A, Auxier J A, Lushbaugh C C. The Importance of Dosimetry tothe Medical Management of Persons Exposed to High Levels of Radiation.In Personal Dosimetry for Radiation Accidents. Vienna: InternationalAtomic Energy Agency; 1965, the contents of which are herebyincorporated by reference). In some embodiments, the present methods andcompositions pertain to a human patient which presents a thrombocytecount reduction of about 50% within about 24 to about 48 hours. In someembodiments, the present human patients experience thrombocytopenia,anemia, and/or neutropenia. Thrombocytopenia is defined as a plateletcount of below 50,000/μL. For example, thrombocytopenia may becharacterized as grade 1 thrombocytopenia (i.e., platelet count of75,000 to 150,000/μL), grade 2 (i.e., platelet count of 50,000 to<75,000 μL), grade 3 (platelet count of 25,000 to <50,000/μL), or grade4 (i.e., platelet count of below 25,000/μL). Anemia may be diagnosed inmen as having a hemoglobin content of less than 13 to 14 g/dL and inwomen as having a hemoglobin content of 12 to 13 g/dL. For example,anemia is divided into various grades based on hemoglobin levels: grade0 (within normal limits, 12 g/dL); grade 1 (mild, 11.9 to 10 g/dL);grade 2 (moderate, 9.9 to 8 g/dL); grade 3 (serious/severe, 7.9 to 6.5g/dL); and grade 4 (life-threatening, <6.5 g/dL). Neutropenia may bedefined as having an absolute neutrophil count (ANC) of less than 1,500cells/mm³. For example, neutropenia is graded as grade 1 (i.e., ANC of1,500/mm³ or less to more than 2,000/mm³), grade 2 (ANC of 1,000/mm³ orless to more than 1,500/mm³), grade 3 (ANC of 500/mm³ or less to morethan 1,000/mm³), or grade 4 (ANC of less than 500/mm³). In variousembodiments, the present methods and compositions reduces the durationand severity of thrombocytopenia, anemia, and/or neutropenia in apatient following irradiation. For example, the present methods andcompositions may reduce the duration and severity of Grade 4thrombocytopenia, anemia, and/or neutropenia in a patient followingirradiation.

In various embodiments, the high dose of radiation refers to a wholebody dose. In various embodiments, the high dose of radiation may not beuniform. In various embodiments, the ARS is a result of a high dose ofradiation. In various embodiments, the high dose of radiation is about 2Gy, or about 2.5 Gy, or about 3 Gy, or about 3.5 Gy, or about 4 Gy, orabout 4.5 Gy, or about 5 Gy, or about 10 Gy, or about 15 Gy, or about 20Gy, or about 25 Gy, or about 30 Gy. In various embodiments, the highdose of radiation is about 5 to about 30 Gy, or about 10 to 25 Gy, orabout 15 to 20 Gy. In some embodiments, the high dose of radiation isassessed by one or more of physical dosimetry and/or biologicaldosimetry (e.g. multiparameter dose assessments), cytogenics (e.g.chromosomal analysis for, for example, blood samples (including, by wayof non-limiting example, dicentric analysis).

In various embodiments, whole-body radiation doses can be divided intosublethal (<2 Gy), potentially lethal (2-10 Gy), and supralethal (>10Gy).

The radiation exposure status (RES) of a given unit is based on theoperational exposure above normal background radiation. It is designedto be an average, based upon unit-level dosimeters. In variousembodiments, the high dose of radiation is sufficient for aclassification of Unit Radiation Exposure Status of RES 3.

In various embodiments, the radiation is ionizing radiation (e.g. one ormore of alpha particles, beta particles, gamma rays, and neutrons) Invarious embodiments, when radiation interacts with atoms, energy isdeposited, resulting in ionization (electron excitation). Thisionization may damage certain critical molecules or structures in a cellby direct and indirect action. The radiation may directly hit aparticularly sensitive atom or molecule in the cell. The damage fromthis is irreparable; the cell either dies or is caused to malfunction.The radiation also can damage a cell indirectly by interacting withwater molecules in the body. The energy deposited in the water leads tothe creation of unstable, toxic hyperoxide molecules; these then damagesensitive molecules and afflict subcellular structures.

In some embodiments, the radiation may be caused by one or more of thefollowing radioactive materials: Americium (e.g. ²⁴¹Am), Cesium (e.g.137Cs), Cobalt (e.g. 60 Co), Uranium (e.g. depleted Uranium), Iodine(e.g. ^(131, 132, 134, 135)I), Phosphorus (e.g. ³²P), Plutonium (e.g.^(238,239)Pu), Radium (e.g. ²²⁶Ra), Strontium (e.g. ⁹⁰Sr), Tritium (e.g.³H), and Uranium (e.g. ^(235, 238, 239)U).

In various embodiments, the high dose of radiation is the result of aradiation disaster. In various embodiments, the human patient is beenexposed or is at risk of being exposed to a high dose of radiation,which may be a result of one or more of a military operation or a firstresponder operation in a contaminated area; a nuclear explosion; acriticality accident; a radiotherapy accident; a terrorist attack;exposure from space travel; escape of radioactive waste; exposure toopen source radiation; and a nuclear reactor malfunction.

In some embodiments, the present methods and compositions find use inaccordance with military operations and/or are suitable for militaryoperations. In some embodiments, the present methods and compositionsfind use in accordance with U.S. Field Manual (FM) 3-11.3, “MULTISERVICETACTICS, TECHNIQUES, AND PROCEDURES FOR CHEMICAL, BIOLOGICAL,RADIOLOGICAL, AND NUCLEAR CONTAMINATION AVOIDANCE,” the contents ofwhich are hereby incorporated by reference in their entirety.

In some embodiments, CBLB502 at the doses and regimens described hereinmay be used in combination with one or more additional agents. In someembodiments, CBLB502 at the doses and regimens described herein may beused in a human patient undergoing treatment with one or more additionalagent. In some embodiments, CBLB502 is used as an adjuvant orneoadjuvant to any of the additional agents described herein.

Adjuvant therapy, also called adjuvant care, is treatment that is givenin addition to the primary, main or initial treatment. By way ofnon-limiting example, adjuvant therapy may be an additional treatmentusually given after primary care where there remains a statistical riskof relapse. In certain embodiments, neoadjuvant therapy refers totherapy to provide a beneficial effect prior to any primary care.

In various embodiments, the additional agents of the present inventioninclude one or more of blood products, colony stimulating factors,cytokines and/or growth factors, antibiotics, diluting and/or blockingagents, mobilizing or chelating agents, stem cell transplants,antioxidants or free radicals, and radioprotectants.

In some embodiments, the blood product is one or more of hematopoieticgrowth factors, such as filgrastim (e.g. NEUPOGEN), a granulocytecolony-stimulating factor (G-CSF), which may be optionally pegylated(e.g. NEULASTA); sargramostim (LEUKINE); and a granulocyte-macrophagecolony-stimulating factor (GM-CSF) and a KSF.

In some embodiments, the additional agent is one or more cytokinesand/or growth factors that may confer radioprotection by replenishingand/or protecting the radiosensitive stem cell populations.Radioprotection with minimal side effects may be achieved by the use ofstem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1fragment IL-1b-rd. Protection may be achieved through induction ofproliferation of stem cells (e.g. via all mentioned cytokines), andprevention of their apoptosis (e.g. via SCF). The treatment allowsaccumulation of leukocytes and their precursors prior to irradiationthus enabling quicker reconstitution of the immune system afterirradiation. SCF efficiently rescues lethally irradiated mice with adose modifying factor (DMF) in range 1.3-1.35 and is also effectiveagainst gastrointestinal syndrome. Flt-3 ligand also provides strongprotection in mice and rabbits.

Several factors, while not cytokines by nature, stimulate theproliferation of the immunocytes and may be used in combination withCBLB502 at the doses and regimens described herein. For example, 5-AED(5-androstenediol) is a steroid that stimulates the expression ofcytokines and increases resistance to bacterial and viral infections.Synthetic compounds, such as ammonium tri-chloro(dioxoethylene-O,O′-)tellurate (AS-101), may also be used to induce secretion of numerouscytokines and for combination with CBLB502. Growth factors and cytokinesmay also be used to provide protection against the gastrointestinalsyndrome. Keratinocyte growth factor (KGF) promotes proliferation anddifferentiation in the intestinal mucosa, and increases thepost-irradiation cell survival in the intestinal crypts. Hematopoieticcytokine and radioprotectant SCF may also increase intestinal stem cellsurvival and associated short-term organism survival.

In certain embodiments, CBLB502 may be added to a regimen of cytokines(e.g. for FILGRASTIM (G-CSF) 2.5-5 μg/kg/d QD s.c. (100-200 μg/μ²/d);for SARGRAMOSTIM (GM-CSF) 5-10 μg/kg/d QD s.c. (200-400 μg/m²/d); and/orfor PEGFILGRASTIM (pegG-CSF) 6 mg once s.c.).

In some embodiments, the additional agent is an interleukin, such asIL-12 (e.g. HEMAMAX (NEUMEDICINES, INC.)).

In some embodiments, the antibiotic is one or more of an anti-bacterial(anti-gram positive and anti-gram negative agents), and/or anti-fungal,and/or anti-viral agent. By way of non-limiting example, in someembodiments, the antibiotic may be a quinolone, e.g. ciprofloxacin,levofloxacin, a third- or fourth-generation cephalosporin withpseudomonal coverage: e.g., cefepime, ceftazidime, or an aminoglycoside:e.g. gentamicin, amikacin, penicillin or amoxicillin, acyclovir,vanomycin. In various embodiments, the antibiotic targets Pseudomonasaeruginosa.

In some embodiments, the additional agent is a diluting and/or blockingagents. For example, stable iodide compounds may be used (e.g. liquid(ThyroShield) and the tablet (losat) KI (NUKEPILLS), Rad Block,I.A.A.A.M., No-Rad, Life Extension (LEF), K14U, NukeProtect, ProKI)). A130 mg dose of daily of oral potassium iodide (KI) may be used inconjunction with CBLB502.

In some embodiments, the additional agent is a mobilizing or chelatingagent. Illustrative mobilizing agents include propylthiouracil andmethimazole, with may reduce the thyroid's retention of radioactivecompounds. Further CBLB502 can be used alongside increasing oral fluidsto a human patient to promote excretion.

Illustrative chelating agents are water soluble and excreted in urine.Illustrative chelating agents include DTPA and EDTA. Dimercaprol formsstable chelates with mercury, lead, arsenic, gold, bismuth, chromium,and nickel and therefore may be considered for the treatment of internalcontamination with the radioisotopes of these elements. Penicillaminechelates copper, iron, mercury, lead, gold, and possibly other heavymetals.

In some embodiments, the additional agent is a stem cell transplant(e.g. bone marrow transplant, PBSCT, MSCT). In some embodiments the stemcell transplant is Remestemcel-L (Osiris) of CLT-008 (Cellerant).

In some embodiments, the additional agent is an antioxidant or freeradical. Antioxidants and free radical scavengers that may be used inthe practice of the invention include, but are not limited to, thiols,such as cysteine, cysteamine, glutathione and bilirubin; amifostine(WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such asIndian holy basil (Ocimum sanctum), orientin and vicenin.

In some embodiments, the additional agent may be a radioprotectant e.g.an antioxidant (e.g. amifostine and vitamin E, gamma tocotrienol (avitamin-E moiety), and genistein (a soy byproduct)), a cytokine (e.g. astem cell factor), a growth factor (e.g. keratinocyte growth factor), asteroid (e.g. 5-androstenediol), ammoniumtrichloro(dioxoethylene-O,O′)tellurate, thyroid protecting agents (e.g.Potassium iodide (KI) or potassium iodate (KlO₃) (e.g. liquid(ThyroShield) and the tablet (losat) KI (NUKEPILLS), Rad Block,I.A.A.A.M., No-Rad, Life Extension (LEF), K14U, NukeProtect, ProKI)),anti-nausea agents, anti-diarrhea agents, antiemetics ((e.g. oralprophylactic antiemetics) such as granisetron (KYTRIL), ondansetron(ZOFRAN), and 5-HT3 blockers with or without dexamethasone), analgesics,anxiolytics, sedatives, cytokine therapy, and antibiotics.

Gastric lavage and emetics, which can be used as additional agents, canbe used to empty the stomach promptly and completely after the ingestionof poisonous materials. Purgatives, laxatives, and enemas, which alsocan be used as additional agents, can reduce the residence time ofradioactive materials in the colon. Further additional agents includeion exchange resins which may limit gastrointestinal uptake of ingestedor inhaled radionuclides, ferric ferrocyanide (Prussian blue) andalginates, which have been used in humans to accelerate fecal excretionof cesium-137.

In still other embodiments, the additional agent may be an agent used totreat radiation-related disorders, such as, for example, 5-AED(Humanetics), Ex-RAD (Onconova), Beclometasone Dipropionate (Soligenix),detoxified endotoxin, EA-230 (Exponential Biotherapies), ON-01210.Na(Onconova), Sothrombomodulin alfa (PAION), Remestemcel-L (Osiris),BIO-100, BIO-200, BIO-300, BIO-400, BIO-500 (Humanetics), CLT-008(Cellerant), EDL-2000 (RxBio), Homspera (ImmuneRegen), MnDTEIP (AeolusPharmaceuticals), RLIP-76 (Terapio), and RX-100 and RX 101 (RxBio).

Further, in some embodiments, CBLB502 (and/or additional agents) can beused in combination with shielding; reduction of radiation exposuretime; and use of agents to reduce body exposure (e.g. uses of gloves,face mask, hood, protective clothing (e.g. anticontamination suits suchas TYVEK ANTI-C SUITS or MOPP-4)).

CBLB502 (and/or additional agents) described herein can possess asufficiently basic functional group, which can react with an inorganicor organic acid, or a carboxyl group, which can react with an inorganicor organic base, to form a pharmaceutically acceptable salt. Apharmaceutically acceptable acid addition salt is formed from apharmaceutically acceptable acid, as is well known in the art. Suchsalts include the pharmaceutically acceptable salts listed in, forexample, Journal of Pharmaceutical Science, 66, 2-19 (1977) and TheHandbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, whichare hereby incorporated by reference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompositions of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Further, CBLB502 (and/or additional agents) described herein can beadministered to a human patient as a component of a composition thatcomprises a pharmaceutically acceptable carrier or vehicle. Suchcompositions can optionally comprise a suitable amount of apharmaceutically acceptable excipient so as to provide the form forproper administration.

Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be, for example, saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea and thelike. In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be used. In one embodiment, the pharmaceuticallyacceptable excipients are sterile when administered to a human patient.Water is a useful excipient when any agent described herein isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid excipients,specifically for injectable solutions. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

The present invention includes the described CBLB502 (and/or additionalagents) in various formulations. CBLB502 (and/or additional agents)described herein can take the form of solutions, suspensions, emulsion,drops, tablets, pills, pellets, capsules, capsules containing liquids,powders, sustained-release formulations, suppositories, emulsions,aerosols, sprays, suspensions, or any other form suitable for use. Inone embodiment, the composition is in the form of a capsule (see, e.g.,U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceuticalexcipients are described in Remington's Pharmaceutical Sciences1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated hereinby reference.

Where necessary, CBLB502 (and/or additional agents) can also include asolubilizing agent. Also, the agents can be delivered with a suitablevehicle or delivery device as known in the art. Combination therapiesoutlined herein can be co-delivered in a single delivery vehicle ordelivery device. Compositions for administration can optionally includea local anesthetic such as, for example, lignocaine to lessen pain atthe site of the injection.

The formulations comprising CBLB502 (and/or additional agents) of thepresent invention may conveniently be presented in unit dosage forms, asfurther described herein, and may be prepared by any of the methods wellknown in the art of pharmacy. Such methods generally include the step ofbringing the therapeutic agents into association with a carrier, whichconstitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g., wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art)

In one embodiment, CBLB502 (and/or additional agents) described hereinis formulated in accordance with routine procedures as a compositionadapted for a mode of administration described herein (e.g. injection,for example, intramuscular injection).

Routes of administration include, for example: intramuscular,intradermal, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically, particularly to theears, nose, eyes, or skin. In some embodiments, the administering iseffected orally or by parenteral injection. The mode of administrationcan be left to the discretion of the practitioner and/or human patient(e.g. in the case of emergency use). In most instances, administrationresults in the release of any agent described herein into thebloodstream.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

CBLB502 (and/or additional agents) described herein can also beadministered orally. CBLB502 (and/or additional agents) can also beadministered by any other convenient route, for example, by intravenousinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and can be administered together with another biologically activeagent. Administration can be systemic or local. Various delivery systemsare known, e.g., encapsulation in liposomes, microparticles,microcapsules, capsules, etc., and can be used to administer.

In one embodiment, CBLB502 (and/or additional agents) described hereinis formulated in accordance with routine procedures as a compositionadapted for oral administration to humans. Compositions for oraldelivery can be in the form of tablets, lozenges, aqueous or oilysuspensions, granules, powders, emulsions, capsules, syrups, or elixirs,for example. Orally administered compositions can comprise one or moreagents, for example, sweetening agents such as fructose, aspartame orsaccharin; flavoring agents such as peppermint, oil of wintergreen, orcherry; coloring agents; and preserving agents, to provide apharmaceutically palatable preparation. Moreover, where in tablet orpill form, the compositions can be coated to delay disintegration andabsorption in the gastrointestinal tract thereby providing a sustainedaction over an extended period of time. Selectively permeable membranessurrounding an osmotically active driving CBLB502 (and/or additionalagents) described herein are also suitable for orally administeredcompositions. In these latter platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving compound, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be useful. Oral compositions can includestandard excipients such as mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, and magnesium carbonate.

In one embodiment, the excipients are of pharmaceutical grade.Suspensions, in addition to the active compounds, may contain suspendingagents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth,etc., and mixtures thereof.

The dosage of any additional agent described herein as well as thedosing schedule can depend on various parameters, including, but notlimited to, the human patient's general health, and the administeringphysician's and/or human patient's discretion. Any additional agentdescribed herein, can be administered prior to (e.g., about 5 minutes,about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24hours, about 48 hours, about 72 hours, about 96 hours, about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6weeks, 8 weeks, or about 12 weeks before), concurrently with, orsubsequent to (e.g., about 5 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours,about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeksafter) the administration of CBLB502, to a human patient in needthereof. In various embodiments any agent described herein isadministered about 1 minute apart, about 10 minutes apart, about 30minutes apart, less than about 1 hour apart, about 1 hour apart, about 1hour to about 2 hours apart, about 2 hours to about 3 hours apart, about3 hours to about 4 hours apart, about 4 hours to about 5 hours apart,about 5 hours to about 6 hours apart, about 6 hours to about 7 hoursapart, about 7 hours to about 8 hours apart, about 8 hours to about 9hours apart, about 9 hours to about 10 hours apart, about 10 hours toabout 11 hours apart, about 11 hours to about 12 hours apart, no morethan about 24 hours apart or no more than about 48 hours apart.

The dose of CBLB502 is disclosed herein. In general, the dose of anyadditional agent that is useful is known to those in the art. Forexample, doses may be determined with reference Physicians' DeskReference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), thecontents of which are incorporated by reference in its entirety. In someembodiment, the present invention allows a patient to receive doses thatexceed those determined with reference Physicians' Desk Reference.

The dosage of any additional agent described herein can depend onseveral factors including the severity of the condition, whether thecondition is to be treated or prevented, and the age, weight, and healthof the human patient to be treated. Additionally, pharmacogenomic (theeffect of genotype on the pharmacokinetic, pharmacodynamic or efficacyprofile of a therapeutic) information about a particular human patientmay affect dosage used. Furthermore, the exact individual dosages can beadjusted somewhat depending on a variety of factors, including thespecific combination of the agents being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the particular disease beingtreated, the severity of the disorder, and the anatomical location ofthe disorder. Some variations in the dosage can be expected.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).

CBLB502 and/or additional agents described herein can be administered bycontrolled-release or sustained-release means or by delivery devicesthat are well known to those of ordinary skill in the art. Examplesinclude, but are not limited to, those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533;5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and5,733,556, each of which is incorporated herein by reference in itsentirety. Such dosage forms can be useful for providing controlled- orsustained-release of one or more active ingredients using, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled- orsustained-release formulations known to those skilled in the art,including those described herein, can be readily selected for use withthe active ingredients of the agents described herein. The inventionthus provides single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled- or sustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

The dosing regimen of CBLB502 is disclosed herein. The dosage regimenfor any additional agent described herein can be selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the human patient; the severity of the condition tobe treated; the route of administration; the renal or hepatic functionof the human patient; the pharmacogenomic makeup of the individual; andthe specific compound of the invention employed. CBLB502 (and/oradditional agents) described herein can be administered in a singledaily dose, or the total daily dosage can be administered in divideddoses of two, three or four times daily. Furthermore, CBLB502 (and/oradditional agents) described herein can be administered continuouslyrather than intermittently throughout the dosage regimen.

In some embodiments, the human patient is a pediatric human. In otherembodiments, the human patient is an adult human. In other embodiments,the human patient is a geriatric human. As females tend to be moretolerant to radiation, in some embodiments, the human patient is a male.

The invention also provides kits that can simplify the administration ofCBLB502 and/or any additional agent described herein. An exemplary kitof the invention comprises CBLB502 and/or any additional agent describedherein in unit dosage form. In one embodiment, the unit dosage form is acontainer, such as a pre-filled syringe, which can be sterile,containing any agent described herein and a pharmaceutically acceptablecarrier, diluent, excipient, or vehicle. The kit can further comprise alabel or printed instructions instructing the use of any agent describedherein. The kit may also include a lid speculum, topical anesthetic, anda cleaning agent for the administration location. The kit can alsofurther comprise one or more additional agent described herein. In oneembodiment, the kit comprises a container containing an effective amountof CBLB502 as disclosed herein and an effective amount of anothercomposition, such as an additional agent as described herein.

In one aspect, the present invention provides a kit suitable for useupon exposure to a high dose of radiation, comprising CBLB502 in a unitdosage form. In some embodiments, the kit comprises CBLB502 in a unitdosage form of about 1 μg to about 50 μg (e.g. about 1, or about 3, orabout 5, or about 10, or about 15, or about 20, or about 25, or about30, or about 35, or about 40, or about 45, or about 45 μg). In someembodiments, the kit further comprises an injection needle (e.g. in aunit dose form, e.g. a pre-loaded (a.k.a. pre-dosed or pre-filled)syringe or a pen needle injector (injection pen)). In some embodiments,the kit comprises CBLB502 which is formulated for intramuscularinjection. In some embodiments, the kit comprises CBLB502 (and/or anyadditional agent) in about 1 to about 3 unit doses. In some embodiments,the present kits further comprise ibuprofen and/or a bottle of water fororal hydration.

In various embodiments, the kit is suitable for military fieldoperations. In various embodiments, the kit further comprises one ormore of a radioactivity detector (e.g. a Geiger counter, ANNDR-2,AN/PDR-77, ADM-300S or a similar device), potassium iodide (KI) orpotassium iodate (KlO₃) (e.g. liquid (ThyroShield) and the tablet(losat) KI (NUKEPILLS), Rad Block, I.A.A.A.M., No-Rad, Life Extension(LEF), K14U, NukeProtect, ProKI; doses of which may be about 130 mg),gloves, face mask, hood, and cleaning solutions, optionally comprisinghypochlorite; and cleaning wipes.

Definitions

The following definitions are used in connection with the inventiondisclosed herein. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofskill in the art to which this invention belongs.

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “additional agent” as used herein is refers to any agent describedherein in addition to CBLB502. As used herein, an additional agentrefers to an agent that can be used in a combination therapy withCBLB502 or the CBLB502 is administered to a human patient that isundergoing treatment with an additional agent.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

Generally, for administering therapeutic agents (e.g. CBLB502 (and/oradditional agents) described herein) for therapeutic purposes, thetherapeutic agents are given at a pharmacologically effective dose. A“pharmacologically effective amount,” “pharmacologically effectivedose,” “therapeutically effective amount,” or “effective amount” refersto an amount sufficient to produce the desired physiological effect oramount capable of achieving the desired result, particularly fortreating the disorder or disease. An effective amount as used hereinwould include an amount sufficient to, for example, delay thedevelopment of a symptom of the disorder or disease, alter the course ofa symptom of the disorder or disease (e.g., slow the progression of asymptom of the disease), reduce or eliminate one or more symptoms ormanifestations of the disorder or disease, and reverse a symptom of adisorder or disease. Therapeutic benefit also includes halting orslowing the progression of the underlying disease or disorder,regardless of whether improvement is realized. Effective amounts ofCBLB502 are disclosed herein.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Non-Human Primate (NHP)Dosing Study

Healthy adult, male or nonpregnant female rhesus macaques wererandomized to receive a single intramuscular dose of 0.0 (n=40), 0.3(n=20), 1.0 (n=19), 3.0 (n=20), 6.6 (n=20), 10 (n=20), 40 (n=20), or 120(n=20) μg/kg of CBLB502 (Entolimod) 25 hours after irradiation (7.2 Gyof total body irradiation). No transfusions, systemic antibiotics,fluids, or hematopoietic growth factors were provided.

FIG. 2 shows a 60-day survival dose-dependent study in non-humanprimates. CBLB502 increased 60-day survival in a dose-dependent manner.The maximal survival benefit was at 10 μg/kg, plateauing across CBLB502doses of 10 μg/kg (adjusted P=0.0021), 40 μg/kg (adjusted P<0.0001), and120 μg/kg (nominal P<0.0001). Sixty-day survival was 27.5% in theplacebo group and 75.0% in the 10 μg/kg dose group. These data suggest,inter alia, that the effective dose of CBLB502 in non-human primates isgreater than at least 3.0 mg/kg.

Example 2: Mitigation of Lethal Acute Radiation Syndrome in Non-HumanPrimates

Methods:

CBLB502 (aka Entolimod) was expressed in E. coli and purified to >98%purity (at SynCo BioPartners, LLC, Amsterdam, The Netherlands) using avalidated cGMP process involving 2-step (ion-exchange and hydrophobicinteraction) chromatographic purification followed by endotoxin removalwith a dedicated ion exchange column. Release testing indicates <100EU/mg endotoxin, <5 ng/mg residual DNA, and <100 ng/mg host cell proteincontent in the entolimod drug product. Absence of additionalcontaminating TLR ligands was confirmed using specific TLR-expressingreporter cell lines (InvivoGen, San Diego, Calif.). The vehicle forentolimod was Dulbecco's Phosphate-Buffered Saline (PBS; Gibco BRL, LifeTechnologies Inc., Grand Island, N.Y.) in earlier studies and PBS-0.1%Tween 80 (O'Brien Pharmacy, Mission, Kans.) in later studies. Animalsreceived a single injection of entolimod or vehicle in the quadricepsmuscle, using a dose volume of 0.2 ml/kg, at 1, 4, 16, 25 or 48 hoursafter the end of irradiation.

For these studies, 2-5 year old rhesus macaques of both genders thatweighed between 3 and 7 kg were used. The animals were research andirradiation naïve, clinically healthy, and certified to be free ofspecific pathogenic microorganisms (such as Salmonella sp., Shigellasp., Mycobacterium tuberculosis, cercopithecine herpesvirus type I (Bvirus), and Toxoplasma gondii). All animals received helminthicidetreatment at their breeding facilities. The care and use of nonhumanprimates (Macaca mulatta, Chinese subspecies, from Sichuan Province)were in accordance with the principles outlined in the current Guide forthe Care and Use of Laboratory Animals published by the NationalInstitutes of Health, and the recommendations of the Weatherall reportfor The Use of Non-Human Primates in Research (December 2006).

Approximately equal numbers of male and female animals were included ineach study group. During the entire observation period (before and afterirradiation), animals were housed in individual stainless steel cages inenvironment-controlled rooms with room temperature of 16-29° C.,relative humidity of 30-70%, and a 12 hour light/dark cycle. In additionto fresh fruits and vegetables daily, animals were fed either primatechow or commercial certified primate biscuits. Fresh drinking water wasprovided ad libitum.

In some irradiation studies, animals received 25 mg/kg pentobarbitalsodium in the animal room to achieve mild anesthesia beforetransportation and an additional 8-10 mg/kg Ketamine injection duringtransit to the irradiation facility. For irradiation, sedated animalswere restrained in plastic irradiation chairs. 5-11 animals wereirradiated simultaneously, with balanced inclusion of animals from allstudy groups in each irradiation cohort. Male and female animals wereirradiated separately. Animals were irradiated bilaterally using thecobalt-60 (60Co) gamma-ray sources located at Sichuan Atomic EnergyInstitute. The first source (used in studies Rs-03, Rs-04, Rs-06, andRs-08) was a vertical bundle of vertically aligned 60Co rods. The secondsource (used in studies Rs-09 and Rs-14) was configured as a verticalrectangular array of vertically aligned 60Co rods. Dose rates indifferent experiments varied from ˜0.8 to 1.1 Gy/min (0.94, 0.92, 0.83,0.73, 1.06 and 1.03 Gy/min for studies Rs-03, Rs-04, Rs-06, Rs-08, Rs-09and Rs-14, respectively) and animals received a total 6.5-6.75 Gy in-airdose (equivalent to 6.0-6.2 Gy midline dose for ˜3-5 kg animals).Individual animal dosimetry was performed using thermoluminescentdosimeter (TLD) sets provided and evaluated by Global DosimetrySolutions, Inc.

In Study Rs-22, animals were sedated with ketamine (10-20 mg/kg) andplaced in plastic restraint boxes for the duration of transport andirradiation. Animals were irradiated using a rotating 6 MV LINAC source(Varian Clinac 2100EX) to a uniform total body in-air dose of 11 Gy, ata dose rate of 0.8±0.025 Gy/min (to achieve a 10.1 Gy midline dose).Dose measurements were made at the center of the cylindrical phantom(diameter=8-10 cm) containing a PTW 31010 0.1 cc Semiflex Ion chamberplaced on the both sides of each animal. NanoDot dosimeters were usedfor measurement of individual surface doses.

All animals participating in studies with survival as the primaryendpoint were observed for 40 days after total body irradiation (TBI).During this period, cage-side observations were performed two or threetimes each day, at least 6 hours apart. Signs of morbidity andmoribundity were recorded. Blood was repeatedly collected from saphenousor cephalic veins for monitoring of complete blood counts (pre-dose,then almost every day on days 1-15, then every 3-4 days) and cytokineand entolimod levels (pre-dose, then at least at 1, 2, 4, 8, 24 hourspost-dose). During blood collection, animals were briefly restrainedwithout sedation. Body weights (at least once per week) and bodytemperature (at least twice per week) were also recorded. Food and fruitconsumption was evaluated daily on a semiquantitative scale (good, fair,or poor). Following irradiation, no intensive individualized supportivecare was provided other than oral rehydration, topical anti-infectivetreatment of lesions and general analgesia with fentanyl patches and/orbuprenorphine when deemed necessary by the study veterinarian. Noketamine or opiates known to affect the immune system were used in thestudy after entolimod treatment. For nutritional support, animals wereprovided with water soaked biscuits and extra amount of fruit.

Moribund animals were subjected to euthanasia based on pre-specifiedcriteria: severe weight loss (>20% loss of initial weight over a 3-dayperiod); complete anorexia (for >3 days, with signs of deterioration);weakness and inability to obtain food or water (for >24 hours); completeunresponsiveness; low core body temperature (<35.9° C.) following aperiod of febrile neutropenia; severe rapidly developing acute anemia(<40 g/L hemoglobin, <13% hematocrit, and a drop of 7% in hematocritbetween consecutive tests); and/or other signs of severe organ systemdysfunction with a poor prognosis (as determined by a veterinarian). Theeuthanasia criteria were based on those generally recommended byveterinarian guidelines for studies involving terminal endpoints withaddition of 2 ARS-specific criteria recommended by Armed ForcesRadiobiology Research Institute (AFRRI), relating to the rapid onset ofacute anemia or drop in core body temperature following a period offebrile neutropenia. Similar criteria for moribund animal euthanasiawere also applied by other groups conducting efficacy studies in the NHPmodel of ARS [27-29, 36, 37]. In studies with survival as the primaryendpoint, all animals that survived to day 40 after TBI were subjectedto scheduled euthanasia. In studies aimed at evaluation ofgastrointestinal (GI) tract morphology, animals were euthanized at 8hours or 5-7 days after TBI (depending on the study). All animals thatwere euthanized or found dead were subjected to gross pathologyexamination, with samples of bone marrow (sternum), spleen, thymus,lymph nodes, and GI tract segments collected for histologicalexamination. In Study Rs-14, bone marrow aspirates were collected fromiliac crests for colony forming assays.

General histological analysis of organ samples was performed by lightmicroscopy of paraffin sections (3-5 μm thick, 1-3 per organ orintestinal segment of each animal) stained with hematoxylin-eosin (H&E).Immunohistochemical detection of SOD2 expression, TUNEL staining and EdUincorporation analyses were performed in deparaffinized sections. Thefollowing antibodies were used for immunohistochemical evaluation: goatanti-SOD2 (N-20) pAb (sc-18503, Santa Cruz Biotechnology, Santa Cruz,Calif.); mouse anti-smooth muscle actin mAb conjugated with Cy3 (C6198,Sigma-Aldrich, St. Louis, Mo.); rabbit anti-phospho-histone H3 pAb(06-570, Millipore, Billerica, Mass.); and rabbit anti-neuro-specifictubulin beta III pAb (ab18207, Abcam, Cambridge, UK). The followingreagents and kits were used histochemical evaluation: ApopTagFluorescent In Situ Apoptosis Detection Kit (S7110, Chemicon, Millipore,Billerica, Mass.) for TUNEL detection, and azide-modified Alexa Fluor488 (Invitrogen, Life Technologies, Grand Island, N.Y.) for EdUincorporation. Samples were examined using a Zeiss Axiolmager A1microscope equipped an epifluorescent light source; images were capturedwith an AxioCam MRc digital camera and processed with a Zeiss AxioImager Z1 microscope (Carl Zeiss, Germany).

The extent of morphologic alterations observed in histological sectionswas assessed by a blind semi-quantitative evaluation.

Analysis of total CFC numbers (which includes CFU-G, CFU-M, CFU-GM,CFU-GEMM, and BFU-E colonies) as well as separate analyses of BFU-E andCFU-Mk were performed using media and reagents from StemCellTechnologies according to the manufacturer's instructions (MethoCult,Cat. #28404; MegaCult-C Cat. #28413; StemCell Technologies, Vancouver,Canada). The number of colonies per 10⁴ live cells was calculated.

Complete blood counts (CBC) analysis was performed using automated bloodcell counters (at Frontier Biosciences: Cell-Dyn 3700SL, Abbott, USA; atUIC TRL: Advia 120, Siemens Healthcare, USA). Cytokine levels in plasma(using K2EDTA as an anticoagulant) were determined using Luminexmultiplex immunological assays at Armed Forces Radiobiology ResearchInstitute (Bethesda, Md.), Baylor Institute for Immunology Research(Dallas, Tex.), or Millipore Corporation (St. Charles, Mo.). In studyRs-03, levels of G-CSF, IL-4, IL-6, IL-10, and IFNγ were analyzed usinghuman-targeted Fluorokine MAP assays from R&D Systems (Minneapolis,Minn.) and levels of IL-2, IL-3, IL-8, IL-12p70, and IP-10 were testedusing Upstate (Temecula, Calif.) human-targeted Beadlyte Multi-CytokineFlex assays. In studies Rs-09 and Rs-14, levels of G-CSF, IL-6, IL-8,and IFNγ were analyzed using Non-Human Primate Cytokine/ChemokineMilliplex Panel from Millipore, Inc. (Billerica, Mass.) and IL-10 wasanalyzed using human-targeted Fluorokine MAP Luminex assay from R&DSystems (Minneapolis, Minn.).

Entolimod levels in serum or plasma were determined using a sandwichELISA method employing proprietary entolimod-specific polyclonalantibodies.

For statistical analysis, the numbers of surviving animals (at 40 daysafter irradiation) were compared pair-wise using Fisher's exact test.Kinetics of mortality was compared between groups by Log rank test. Foranalysis of the effect of entolimod treatment on survival, the naturallogarithm of odds ratio of survival (odds of survival in the treatedgroup divided by that in the control group) was chosen as the metric.The odds associated with a probability p were defined as p/(1−p). For agroup of size n with 100% survival, odds were defined as (n−0.5)/n; forgroups with 0% survival odds were defined as 0.5/n. Quantitative datawere evaluated using Student's t-test. All tests were two-sided.P-values <0.05 were considered statistically significant. Error bars ingraphs represent standard errors (unless specified otherwise). GraphPadPrism 5.0 and Microsoft Excel 2007-2010 were used for most statisticalanalyses.

Calculation of days with Grade 4 cytopenia/anemia: definition of a studyday as cytopenic/anemic was based on actual values when available, or onimputed values for days when samples were not collected. Imputation wasperformed by linear interpolation over time between actual measurements,or by using the last observation carried forward between the day of lastavailable measured value and the day of death. Percentage of live dayswith Grade 4 cytopenia/anemia was calculated as number of days withcytopenia/anemia divided by number of days the animal was alive duringthe 40-day observation period.

Area under the curve (AUC) values for cytokine and entolimod levels werecalculated using the trapezoid rule. To eliminate the influence ofdifferences in basal cytokine levels, AUC values werebackground-adjusted by subtracting the minimum observed factorconcentration from all other concentrations before calculation.

Results:

Treatment of NHPs with entolimod within 48 hours after lethal TBIsignificantly reduces the risk of death from ARS

To investigate the potency of entolimod in increasing survival of NHPswhen administered after lethal TBI, a series of non-GLP studies inrhesus macaques were performed using a TBI dose range ofLD_(50/40)-LD_(75/40) (50-75% lethal over 40 days). This TBI dose rangewas chosen as being an approximate upper threshold at which exposedindividuals would be at substantial risk of death, but might still besalvageable by medical therapy. This study presents the resultsgenerated within four survival experiments, designated Rs-03, Rs-06,Rs-09 and Rs-14, involving a total of 164 animals. The study groups forall 4 experiments are shown in Table 1.

TABLE 1 Efficacy of a single injection of entolimod in increasing 40-daysurvival of lethally irradiated NHPs when administered at different doselevels within 1-48 hours after TBI 40-day survival Kinetics of mortalityInjection Absolute Survival Mean time(s) survival odds survivalIrradiation Entolimod relative Group No. of % of increase P- ratio vs.time ± SE, P- Study dose dose, μg/kg to TBI, h size(n) survivorssurvivors % value^(A) vehicle days value^(B) Rs-03 ~LD_(75/40)     0(vehicle)  +1 10 2 20% — — — 18.7 ± 3.7 — (6.5 Gy)^(C)    40  +1 10 770% 50% 0.07 9.33 30.8 ± 4.8 0.06 Rs-06 ~LD_(75/40)     0 (vehicle) +16 8 2 25% — — — 23.3 ± 3.8 — (6.5 Gy)^(C)    40 +16 12 8 67% 42% 0.176.00 30.6 ± 4.1 0.17    40 +25 10 7 70% 45% 0.15 7.00 35.0 ± 2.7 0.02   40 +48 12 8 67% 42% 0.17 6.00 32.4 ± 3.4 0.10 Rs-09 ~LD_(50/40)     0(vehicle)  +1 18 9 50% — — — 29.4 ± 2.7 — (6.75 Gy)^(D)     0.3  +1 1812  67% 17% 0.50 2.00 32.0 ± 2.7 0.44     3  +1 18 14  78% 28% 0.16 3.5035.7 ± 2.0 0.07    10  +1 18 17  94% 44% 0.007 17.00  39.2 ± 0.8 0.003Rs-14 ~LD_(50/40)     0 (vehicle) +25 10 4 40% — — — 24.5 ± 4.3 — (6.75Gy)^(D)    10 +25 10 10  100%  60% 0.01 28.50 ^(G) 40.0 ± 0.0 0.004   40 +25 10 8 80% 40% 0.17 6.00 35.3 ± 3.1 0.06 Pooled vehicle~LD_(50-75/40)     0 (vehicle)^(E) +1-+25 46 17  37% — — — 24.9 ± 1.8 —vs. ≥10 μg/kg (6.5-6.75 Gy) ≥10^(F) +25 30 25  83% 46% 0.0001 8.53 36.8± 1.4 0.0001 entolimod, +25 h ^(A)P-value by Fisher's exact test(two-tailed) for comparisons vs. vehicle groups within individualstudies or in pooled group analysis ^(B)P-value by Log rank test(two-tailed) for comparisons vs. vehicle groups within individualstudies or in pooled group analysis ^(C)Source I: Sichuan Atomic EnergyInstitute, cylindrical bundle of Co-60 rods ^(D)Source II: SichuanAtomic Energy Institute, vertical array of Co-60 rods^(E)Vehicle-treated animals from studies Rs-03, Rs-06, Rs-09, and Rs-14^(F)Entolimod-treated animals from studies Rs-06 and Rs-14 ^(G)Survivalodds and survival odds ratios adjusted due to 100% survival are shown initalics Note: The frequency of moribund euthanasia was as follows: StudyRs-03 - 91% (1/11 - found dead), Study Rs-06 - 100%; Study Rs-09 - 85%(3/20 - found dead); Rs-14 - 88% (1/8 - found dead). The likely cause ofdeath in all the non-euthanized animals was acute hemorrhage.

In all of these studies, the effects of intramuscular (i.m.) injectionof entolimod were monitored for 40 days after irradiation. In additionto monitoring animal morbidity and mortality, multiple physiologicalparameters, blood cell counts, levels of elicited cytokines inperipheral blood (pharmacodynamics) and entolimod pharmacokinetics (PK)were evaluated. To prevent suffering, moribund animals were euthanizedaccording to a predefined set of criteria (uniformly used in NHP studiesdescribed here). No supportive care was provided other than oralrehydration (drinking water given ad libitum) and non-systemic treatmentof external lesions. Study groups were composed of approximately equalnumbers of male and female animals. Following irradiation, animalsgenerally developed a clinical picture of ARS with typical prodromal andmanifest illness features.

First, in studies Rs-03 and Rs-06, the effective timeframe forradiomitigative efficacy of 40 μg/kg entolimod (a dose previouslyestablished as radioprotective in NHPs) was examined. Animals (n=8-12per group) were irradiated with 6.5 Gy TBI and treated with entolimod at1, 16, 25, or 48 hours after irradiation. Forty-day survival invehicle-treated groups was 20% and 25% in studies Rs-03 and Rs-06,respectively, whereas survival in all entolimod-treated groups was67-75%. Thus, NHP survival was improved by an absolute 42-50% withsurvival odds ratios ranging from 6 to 9.3 regardless of entolimodadministration time within the first 48 hours after TBI (FIG. 3, panelsA and B; and Table 1).

The two subsequent studies, Rs-09 and Rs-14, explored dose-dependence ofthe survival effect of entolimod in NHPs at the boundaries of the25-hour post-TBI period with the drug injected at either 1 or 25 hoursafter TBI, respectively. Animals (n=10-18 per group) were irradiatedwith 6.75 Gy TBI (from a different radiation source compared to the twofirst studies) and received vehicle or entolimod injections at 0.3, 3 or10 μg/kg (Rs-09) or 10 or 40 μg/kg (Rs-14) dose levels. Forty-daysurvival in vehicle-treated groups was 50% (Rs-09) or 40% (Rs-14), whileentolimod doses of 10-40 μg/kg were fully efficacious in rescuing80-100% of irradiated NHPs. These data correspond to increases insurvival of 40-60% with survival odds ratios ranging between 6 and 28.5.Entolimod treatment at 3 μg/kg provided partial efficacy (28% survivalincrease) and the lowest tested dose of 0.3 μg/kg showed little or noefficacy (17% survival increase) (FIG. 3, panels C and D; and Table 1).

The survival advantage of 40-60% provided by efficacious entolimod doseswas uniformly observed across all four of the studies reported here,although statistical significance was not reached in some individualgroups (probably due to their small size). Similarity of study designelements, treatment regimens, and animal populations allowedmeta-analysis in which similarly treated groups were pooled. Pooling ofvehicle-treated groups from all 4 studies (n=46) resulted in 37% 40-daysurvival, while pooling of all groups treated with fully efficacious 10and 40 μg/kg entolimod doses at 25 hours after TBI (n=30) indicated 83%40-day survival. This 46% increase in 40-day survival was highlystatistically significant (P=0.0001 by Fisher's exact test) with asurvival odds ratio of 8.5 (Table 1).

The kinetics of mortality was similar in all 4 studies, with themajority of deaths occurring on days 12-16 after TBI and no deathsoccurring after day 30. Mean survival time ranged from 18.7±3.7 to29.4±2.7 days in vehicle-treated groups (24.9±1.8 days with allvehicle-treated groups pooled), while after 10 μg/kg entolimodtreatment, mean survival time substantially increased to a range of30.6±4.1 to 40.0±0.0 days (36.8±1.4 days in pooled group analysis;P=0.0001 by Log-rank test for difference in survival kinetics) (FIG. 3and Table 1).

Entolimod Treatment Accelerates Recovery of Hematopoiesis in LethallyIrradiated NHPs

Radiation damage to the hematopoietic (HP) system is one of the majorcauses of lethality at ˜LD₅₀-LD₇₀ doses of TBI. Therefore, toinvestigate the mechanisms underlying entolimod's radiomitigativeefficacy, the content of different hematopoietic cell types was examinedin peripheral blood and bone marrow samples collected in the four NHPstudies described above.

Comparison of hematology data from control lethally irradiated NHPs andthose treated with a single injection of entolimod revealed that acrossall four studies, the efficacious drug doses of 10 μg/kg reduced theduration and severity of thrombocytopenia, neutropenia, and anemia whengiven at any tested time point within 1-48 hours post-TBI (FIG. 4,panels A-D, G-H; FIG. 5, panels A-D, G-H; Tables 2-6).

TABLE 2 Mean nadir values of neutrophils, platelets and hemoglobin inperipheral blood following total body irradiation and vehicle orentolimod treatment Injection Neutrophils Platelets Hemoglobin time(s)Group Mean Mean Mean Irradiation Entolimod relative size nadir ± SE P-nadir ± SE P- nadir ± SE P- Study dose dose, μg/kg to TBI, h (n)(×10³/μL) value^(A) (×10³/μL) value^(A) (g/L) value^(A) Rs-03~LD_(75/40)     0 (vehicle)  +1 10 0.01 ± 0.005 —   31 ± 27.9 — 59.8 ±7.4 — (6.5 Gy)^(B)    40  +1 10 0.18 ± 0.128 0.20 59.6 ± 26.2 0.46 77.8± 6.9 0.09 Rs-06 ~LD_(75/40)     0 (vehicle) +16  8 0.01 ± 0.011 — 3.2 ±1.7 — 72.5 ± 5.4 — (6.5 Gy)^(B)    40 +16 12 0.06 ± 0.017 0.05   34 ±16.4 0.09 92.9 ± 7.4 0.04    40 +25 10 0.02 ± 0.007 0.51 15.8 ± 5.9 0.07 83.3 ± 5.4 0.18    40 +48 12 0.02 ± 0.005 0.85 12.5 ± 4.6  0.0893.1 ± 3.2 0.01 Rs-09 ~LD_(50/40)     0 (vehicle)  +1 18 0.01 ± 0.001 —  8 ± 1.4 — 66.1 ± 3.7 — (6.75 Gy)^(C)     0.3  +1 18 0.01 ± 0.003 0.447.2 ± 1.2 0.66 69.1 ± 3.5 0.56     3  +1 18 0.02 ± 0.006 0.02 16.6 ±3.7  0.04 78.9 ± 4.1 0.03    10  +1 18 0.03 ± 0.009 0.01 22.4 ± 3.9  0.002 76.7 ± 3.7 0.05 Rs-14 ~LD_(50/40)     0 (vehicle) +25 10 0.01 ±0.006 — 6.8 ± 2.4 — 60.2 ± 7.6 — (6.75 Gy)^(C)    10 +25 10 0.04 ± 0.0110.11 21.8 ± 5.4  0.03 77.5 ± 3.8 0.06    40 +25 10 0.07 ± 0.029 0.0939.9 ± 12.4 0.03 89.6 ± 4.4 0.005 Pooled vehicle ~LD_(50-75/40)     0(vehicle)^(D) +1-+25 46 0.01 ± 0.003 — 11.9 ± 6.1  — 64.6 ± 2.9 — vs.≥10 μg/kg (6.5-6.75 Gy) ≥10^(E) +25 30 0.04 ± 0.011 0.006 25.9 ± 5.1 0.08 83.5 ± 2.7 <0.0001 entolimod, +25 h ^(A)P-value by Student's t-test(two-tailed) for comparisons vs. vehicle groups within individualstudies or in pooled group analysis ^(B)Source I: Sichuan Atomic EnergyInstitute, cylindrical bundle of Co60 rods ^(C)Source II: Sichuan AtomicEnergy Institute, vertical array of Co60 rods ^(D)Vehicle-treatedanimals from studies Rs-03, Rs-06, Rs-09, and Rs-14^(E)Entolimod-treated animals from studies Rs-06 and Rs-14

TABLE 3 S1 Table. Incidence and duration of Grade 4 neutropenia(neutrophil count <500 cells/μL) in lethally irradiated NHPs treatedwith vehicle or entolimod Injection Group Mean % live days ± Incidenceof Entolimod time(s) relative size SE with Grade 4 P- Grade 4 P- StudyIrradiation dose dose, μg/kg to TBI, h (n) neutropenia value^(A)neutropenia value^(B) Rs-03 ~LD_(75/40)     0 (vehicle)  +1 10 56% ± 6%— 100% — (6.5 Gy)^(C)    40  +1 10 33% ± 6% 0.01  90% >0.05 Rs-06~LD_(75/40)     0 (vehicle) +16  8 60% ± 6% — 100% — (6.5 Gy)^(C)    40+16 12 47% ± 6% 0.13 100% >0.05    40 +25 10 44% ± 6% 0.08 100% >0.05   40 +48 12 51% ± 8% 0.35 100% >0.05 Rs-09 ~LD_(50/40)     0 (vehicle) +1 18 51% ± 4% — 100% — (6.5 Gy)^(D)     0.3  +1 18 48% ± 5% 0.65100% >0.05     3  +1 18 44% ± 4% 0.22 100% >0.05    10  +1 18 38% ± 2%0.01 100% >0.05 Rs-14 ~LD_(50/40)     0 (vehicle) +25 10 55% ± 6% — 100%— (6.5 Gy)^(D)    10 +25 10 37% ± 2% 0.01 100% >0.05    40 +25 10 42% ±5% 0.11 100% >0.05 Pooled vehicle vs. ~LD_(50-75/40)     0 (vehicle)^(E)+1-+25 46 55% ± 3% — 100% — ≥10 μg/kg (6.5-6.75 Gy) ≥10^(F) +25 30 41% ±3%  0.001 100% >0.05 entolimod, +25 h ^(A)P-value by Student's t-test(two-tailed) against vehicle groups in individual studies or in pooledgroup analysis ^(B)P-value by Fisher's exact test (two-tailed) againstvehicle groups in individual studies or in pooled group analysis^(C)Source I: Sichuan Atomic Energy Institute, cylindrical bundle ofCo-60 rods ^(D)Source II: Sichuan Atomic Energy Institute, verticalarray of Co-60 rods ^(E)Vehicle-treated animals from studies Rs-03,Rs-06, Rs-09, and Rs-14 ^(F)Entolimod-treated animals from studies Rs-06and Rs-14

TABLE 4 S2 Table. Incidence and duration of absolute neutropenia(neutrophil count <10 cells/μL) in lethally irradiated NHPs treated withvehicle or entolimod Injection Group Mean % live days ± Incidence ofIrradiation Entolimod time(s) relative size SE with absolute absoluteStudy dose dose, μg/kg to TBI, h (n) neutropenia P-value^(A) neutropeniaP-value^(B) Rs-03 ~LD_(75/40)     0 (vehicle)  +1 10 16% ± 4%  — 80% —(6.5 Gy)^(C)    40  +1 10 1% ± 1% 0.002  0% 0.001 Rs-06 ~LD_(75/40)    0 (vehicle) +16  8 15% ± 3%  — 88% — (6.5 Gy)^(C)    40 +16 12 3% ±2% 0.01 25% 0.02    40 +25 10 6% ± 2% 0.03 50% >0.05    40 +48 12 8% ±3% 0.11 50% >0.05 Rs-09 ~LD_(50/40)     0 (vehicle)  +1 18 10% ± 2%  —78% — (6.75 Gy)^(D)     0.3  +1 18 11% ± 3%  0.74 72% >0.05     3  +1 187% ± 2% 0.39 50% >0.05    10  +1 18 4% ± 1% 0.02 56% >0.05 Rs-14~LD_(50/40)     0 (vehicle) +25 10 6% ± 2% — 50% — (6.75 Gy)^(D)    10+25 10 2% ± 1% 0.19 30% >0.05    40 +25 10 5% ± 3% 0.84 50% >0.05 Pooledvehicle vs. ~LD_(50-75/40)     0 (vehicle)^(E) +1-+25 46 11% ± 1%  — 74%— ≥10 μg/kg (6.5-6.75 Gy) ≥10^(F) +25 30 4% ± 1% 0.0003 43% 0.01entolimod, +25 h AP-value by Student's t-test (two-tailed) againstvehicle groups in individual studies or in pooled group analysis^(B)P-value by Fisher's exact test (two-tailed) against vehicle groupsin individual studies or in pooled group analysis ^(C)Source I: SichuanAtomic Energy Institute, cylindrical bundle of Co-60 rods ^(D)Source II:Sichuan Atomic Energy Institute, vertical array of Co-60 rods^(E)Vehicle-treated animals from studies Rs-03, Rs-06, Rs-09, and Rs-14^(F)Entolimod-treated animals from studies Rs-06 and Rs-14

TABLE 5 S3 Table. Incidence and duration of Grade 4 thrombocytopenia(platelet count <10,000 cells/μL) in lethally irradiated NHPs treatedwith vehicle or entolimod Group Mean % live days ± Entoli-mod Injectiontime(s) size SE with Grade 4 Incidence of Grade 4 Study Irradiation dosedose, μg/kg relative to TBI, h (n) thrombo-cytopenia P-value^(A)thrombo-cytopenia P-value^(B) Rs-03 ~LD_(75/40)     0 (vehicle)  +1 1020% ± 4%  — 80% — (6.5 Gy)^(C)    40  +1 10 2% ± 1% 0.003 20% 0.02 Rs-06~LD_(75/40)     0 (vehicle) +16  8 26% ± 5%  — 88% — (6.5 Gy)^(C)    40+16 12 6% ± 3% 0.003 33% 0.03    40 +25 10 11% ± 4%  0.02 60% >0.05   40 +48 12 13% ± 4%  0.04 58% >0.05 Rs-09 ~LD_(50/40)     0 (vehicle) +1 18 9% ± 2% — 72% — (6.75 Gy)^(D)     0.3  +1 18 9% ± 2% 0.8967% >0.05     3  +1 18 5% ± 2% 0.09 44% >0.05    10  +1 18 3% ± 2% 0.0328% 0.02 Rs-14 ~LD_(50/40)     0 (vehicle) +25 10 20% ± 5%  — 80% —(6.75 Gy)^(D)    10 +25 10 2% ± 1% 0.004 40% >0.05    40 +25 10 4% ± 2%0.01 30% >0.05 Pooled ~LD_(50-75/40)     0 (vehicle)^(E) +1-+25 46 17% ±2%  — 78% — vehicle vs. (6.5-6.75 Gy) ≥10^(F) +25 30 5% ± 2% <0.0001 43%0.003 ≥10 μg/kg entoli-mod, +25 h ^(A)P-value by Student's t-test(two-tailed) against vehicle groups in individual studies or in pooledgroup analysis ^(B)P-value by Fisher's exact test (two-tailed) againstvehicle groups in individual studies or in pooled group analysis^(C)Source I: Sichuan Atomic Energy Institute, cylindrical bundle ofCo60 rods ^(D)Source II: Sichuan Atomic Energy Institute, vertical arrayof Co60 rods ^(E)Vehicle-treated animals from studies Rs-03, Rs-06,Rs-09, and Rs-14 ^(F)Entolimod-treated animals from studies Rs-06 andRs-14

TABLE 6 S4 Table. Incidence and duration of Grade 4 anemia (hemoglobinlevel <65 g/L) in lethally irradiated NHPs treated with vehicle orentolimod Group Mean % live days ± Entolimod Injection time(s) size SEwith Grade 4 Incidence of Study Irradiation dose dose, μg/kg relative toTBI, h (n) anemia P-value^(A) Grade 4 anemia P-value^(B) Rs-03~LD_(75/40)     0 (vehicle)  +1 10 11% ± 3%  — 70% — (6.5 Gy)^(C)    40 +1 10 6% ± 3% 0.29 20% >0.05 Rs-06 ~LD_(75/40)     0 (vehicle) +16  88% ± 5% — 25% — (6.5 Gy)^(C)    40 +16 12 2% ± 2% 0.25  8% >0.05    40+25 10 6% ± 4% 0.78 20% >0.05    40 +48 12 1% ± 1% 0.20  0% >0.05 Rs-09~LD_(50/40)     0 (vehicle)  +1 18 9% ± 3% — 44% — (6.75 Gy)^(D)     0.3 +1 18 7% ± 2% 0.49 33% >0.05     3  +1 18 6% ± 3% 0.50 17% >0.05    10 +1 18 6% ± 3% 0.41 17% >0.05 Rs-14 ~LD_(50/40)     0 (vehicle) +25 1012% ± 4%  — 60% — (6.75 Gy)^(D)    10 +25 10 1% ± 1% 0.01 20% >0.05   40 +25 10 1% ± 1% 0.02 10% >0.05 Pooled ~LD_(50-75/40)     0(vehicle)^(E) +1-+25 46 10% ± 2%  — 50% — vehicle vs. (6.5-6.75 Gy)≥10^(F) +25 30 3% ± 2% 0.003 17% 0.004 ≥10 μg/kg entoli-mod, +25 h^(A)P-value by Student's t-test (two-tailed) against vehicle groups inindividual studies or in pooled group analysis ^(B)P-value by Fisher'sexact test (two-tailed) against vehicle groups in individual studies orin pooled group analysis ^(C)Source I: Sichuan Atomic Energy Institute,cylindrical bundle of Co60 rods ^(D)Source II: Sichuan Atomic EnergyInstitute, vertical array of Co60 rods ^(E)Vehicle-treated animals fromstudies Rs-03, Rs-06, Rs-09, and Rs-14 ^(F)Entolimod-treated animalsfrom studies Rs-06 and Rs-14

In the context of ARS, anemia should be interpreted as a result ofhemorrhage exacerbated by radiation-imposed suppression of compensatingerythropoiesis (FIG. 4, panels E and F; and FIG. 5., panels E and F)since mature erythrocytes are radioresistant and their life span in thecirculation of NHPs is 60 days.

Although small group sizes reduced the statistical significance of theeffects in some individual cases, the positive trends indicatingentolimod-mediated amelioration of HP ARS were clearly observed in allreported studies (Tables 2-6). In pooled group analysis (analogous tothat described for survival), entolimod effects were highlystatistically significant. Thus, nadir neutrophil counts were increasedby entolimod treatment from 0.01±0.003×10³/p1 to 0.04±0.011×10³/p1(P=0.006), nadir platelet counts—from 11.9±6.1×10³/p1 to 25.9±5.1×10³/p1(P=0.08), and nadir hemoglobin levels—from 64.6±2.9×10³/p1 to83.5±2.7×10³/p1 (P<0.0001) (Table 2). At the same time, the proportionof live days (when a particular animal was alive and had cytopenia) withGrade 4 neutropenia (neutrophil counts <500/μl) was decreased byentolimod treatment from 55%±3% in vehicle-treated groups to 41%±3%(P=0.001, Table 3), with Grade 4 thrombocytopenia (platelet counts<10,000/μl)—from 17%±2% to 5%±2% (P<0.0001, Table 5), and with Grade 4anemia (hemoglobin level <65 g/L)—from 10%±2% to 3%±2% (P=0.003, Table6). While entolimod treatment did not change the incidence of Grade 4neutropenia (proportion of animals that developed this condition atleast once during 40 days of observation), it reduced the incidence ofGrade 4 thrombocytopenia from 78% in vehicle-treated groups to 43%(pooled group analysis, P=0.003, Table 5), and of Grade 4 anemia—from50% in control groups to 17% (pooled group analysis, P=0.004, Table 6).In addition to decreased severity and duration of thrombocytopenia,accelerated recovery of erythropoiesis in entolimod-treated NHPs (FIG.4, panels E and F; and FIG. 5, panels E and F) also contributed tomarkedly decreased incidence of ARS-associated Grade 4 anemia. Unlikethe survival endpoint, where saturation of entolimod's effect wasachieved at dose of 10 μg/kg (FIG. 3, panels C and D), the effect ofentolimod on hematological parameters continued to improve between 10and 40 μg/kg doses (FIG. 4, panels B, D, F and H). This is consistentwith the notion that achieving certain threshold levels (for example,above Grade 4 cytopenias/anemia) is sufficient to support survival.

Consistent with the finding of accelerated recovery of blood cellularityafter hematopoietic nadirs in entolimod-treated irradiated NHPs, thebone marrow (BM) of treated animals displayed accelerated morphologicalrecovery. Analysis of hematoxylin-eosin-stained sternum sectionscollected from surviving NHPs at 40 days post-TBI showed that BM fromanimals given a single injection of 40 μg/kg entolimod within 48 hoursafter TBI was considerably better regenerated compared to controlmonkeys. The hematopoietic cells were not only numerous, but alsodensely arranged in clusters among the sinusoids and the inconspicuousfat component. The elements of the three hematopoietic lineages(granulocytic, erythroid, and megakaryocytic) were spread out and inclose contact with each other. In some animals, BM morphology was normalor close to normal, although others still had slightly or moderatelyhypoplastic BM. In contrast, BM of control NHPs was clearly lesscellular and contained more adipose elements. Accelerated morphologicalrecovery in entolimod-treated NHPs was also observed in lymphoid organs,including thymus, spleen and lymph nodes (FIG. 6). These differenceswere statistically significant when blindly assigned semi-quantitativehistological scores were compared (Table 7). Similar effects wereinduced by a single injection of 10 μg/kg entolimod administered at 25hours after TBI (FIG. 7; and Table 8).

TABLE 7 Histological evaluation of hematopoietic/lymphoid organs fromNHPs that survived to day 40 after 6.5 Gy TBI and vehicle or entolimodtreatment (study Rs-06) Mean score ^(A) ± SE P-value vs. vehicle ^(B) —Entolimod, 40 μg/kg Entolimod, Vehicle 16 h 25 h 48 h 40 μg/kgOrgan/tissue (N = 2) (N = 8) (N = 7) (N = 8) 16 h 25 h 48 h Bone marrow1.3 ± 0.3 3.4 ± 0.1 3.6 ± 0.2 3.4 ± 0.2 0.04 0.01 0.02 Thymus 1.0 ± 0.02.4 ± 0.5 2.6 ± 0.5 2.3 ± 0.4 0.02 0.02 0.02 Spleen 1.0 ± 0.0 2.0 ± 0.31.7 ± 0.4 1.5 ± 0.2 0.01 0.14 0.03 Lymph node 1.0 ± 0.0 1.6 ± 0.3 1.9 ±0.3 2.0 ± 0.2 0.05 0.02 0.001 ^(A) Scoring was performed based on a5-grade scale developed for each organ: 0 - total aplasia; 1 -pronounced atrophy, 2 - moderate atrophy, 3 - slight atrophy, close tonormal morphology; 4 - normal morphology. Scoring criteria forindividual organs are described in Supporting Information, S1 Methods.^(B) Student's t-test vs. vehicle, 2-tailed.

TABLE 8 Semi-quantitative histological evaluation of hematopoietic/lymphoid organs from NHPs that survived to day 40 after 6.75 Gy TBIfollowed by vehicle or entolimod treatment (study Rs-14) Mean score ^(A)± SE P-value vs. vehicle ^(B) Entolimod, +25 h Entolimod, +25 h Vehicle10 μg/kg 40 μg/kg 10 40 Organ/tissue (N = 4) (N = 10) (N = 8) μg/kgμg/kg Bone marrow 2.2 ± 0.7 3.7 ± 0.1 3.9 ± 0.0 0.1 0.1 Thymus ^(C) 2.1± 0.1 3.5 ± 0.2 3.8 ± 0.1 0.007 0.002 Spleen 1.0 ± 0.3 2.7 ± 0.2 3.6 ±0.1 0.004 0.001 Lymph node 1.2 ± 0.2 2.3 ± 0.3 3.7 ± 0.1 0.007 0.0003^(A) Scoring was performed based on a 5-grade scale developed for eachorgan: 0-total aplasia; 1-pronounced atrophy, 2-moderate atrophy,3-slight atrophy, close to normal morphology; 4-normal morphology.Scoring criteria for individual organs are described in SupplementaryMethods. ^(B) Student's t-test vs. vehicle, 2-tailed. ^(C) Due to thymusatrophy, fewer thymus samples were evaluated compared to other organs (N= 2, 4, and 6 for vehicle, 10 μg/kg groups, respectively).

To investigate the kinetics of entolimod-elicited BM recovery in moredetail, a dedicated study was performed in which mice were injected withvehicle or entolimod 25 hours after 9 Gy TBI (˜LD_(50/30)) and theneuthanized for histological and other evaluations at different timepoints. As shown in FIG. 8, panel A, the first histological signs ofactive hematopoiesis were evident in the BM of entolimod-treated mice asearly as 3 days after TBI (2 days after drug treatment), with full-scalehematopoiesis observed by day 14. In comparison, the onset ofhematopoietic recovery in vehicle-treated mice occurred betweenpost-irradiation days 14 and 28, and full-scale hematopoiesis wasfurther shifted to a time interval between days 28 and 56.Interestingly, the first signs of hematopoiesis in the BM ofentolimod-treated mice were localized to the trabecular cell lining,suggesting stimulation of the HP stem cell compartment by entolimod.Indeed, as early as 7 days after TBI, very early granulomonocyticprogenitors (CFU-GM colonies) from entolimod-treated mice were elevatedin number and displayed increased proliferative potential compared tothose from vehicle-treated mice (FIG. 8, panel B).

Analysis of BM aspirates obtained from NHPs on day 40 after exposure to6.75 Gy TBI and treated with either vehicle (n=4) or 40 μg/kg entolimod(n=4) 25 hours later revealed a clear positive influence of the drug onthe content of hematopoietic progenitor cells, including total colonyforming cells (CFC), erythroid burst forming units (BFU-E), andmegakaryocyte colony forming units (CFU-Mk) (granulocyte lineageprogenitors were not separately analyzed). The most substantialentolimod-elicited effect was on CFU-Mk, for which the frequency per 10⁴viable BM cells was increased ˜4.8-fold from 0.34±0.11 invehicle-treated animals to 1.63±0.05 (P<0.05). The frequencies of CFCand BFU-E in entolimod-treated animals were increased by 22% and 36%,respectively, compared to vehicle-treated controls. The observeddominance of entolimod's effect on CFU-Mk compared to progenitors ofother lineages at this late time point (40 days after TBI) may be due todifferences in the kinetics of recovery of different HP lineages, withthe thrombopoietic lineage being known for its slow restorationfollowing BM ablation and transplantation compared to other lineages.

Taken together, these data demonstrate, inter alia, that entolimod is apotent mitigator of radiation injury in the HP system, and acts viastimulation of accelerated hematopoietic recovery.

Entolimod Treatment Reduces Initial Damage in the GI Tract andAccelerates its Regeneration in Lethally Irradiated NHPs

Acute high-dose irradiation sufficient to induce GI ARS results in highdegrees of apoptosis in the GI tract mucosa and submucosal elements(lamina propria, mucosa muscularis, lymphoid accumulations), leading toatrophy, increased permeability, susceptibility to hemorrhage(especially on the background of thrombocytopenia) and/orintussusceptions. Histological analyses were used to evaluate theeffects of entolimod treatment on these signs of radiation damage to theNHP GI tract.

Assessment of NHP GI histology at 40 days after LD_(50-75/40) TBI dosesdid not reveal substantial differences between surviving entolimod- orvehicle-treated irradiated animals (Table 9), most likely due tonear-completion of regeneration by this late post-TBI time pointregardless of treatment. Nevertheless, mean histological scores weregenerally higher in entolimod-treated groups compared to vehicle-treatedcontrol groups (Table 9). This radiomitigative/pro-regeneration effectof entolimod was most apparent in the radiosensitive small intestine andwas observed in all histological substructures of the GI tract (villiand/or surface epithelium; crypts; and lamina propria with submucosa).The level of radiomitigation was moderate in the cecum and minimal tononexistent in the colon and rectum, where radiation injury was notprominent (as observed in histological analysis of samples from animalsthat died during the course of the study).

TABLE 9 Semi-quantitative histological evaluation of GI tract segmentsfrom NHPs that survived to day 40 after 6.5 Gy TBI and vehicle orentolimod treatment (study Rs-06) Mean score ^(A) ± SE P-value vs.vehicle ^(B) Entolimod, 40 μg/kg Entolimod, Organ/ Vehicle +16 h +25 h+48 h 40 μg/kg tissue (N = 2) (N = 8) (N = 7) (N = 8) 16 h 25 h 48 hDuodenum 2.4 ± 0.1 3.1 ± 0.2 2.9 ± 0.2 3.2 ± 0.1 0.03 0.07 0.01 Jejunum2.8 ± 0.3 3.8 ± 0.1 3.6 ± 0.1 3.6 ± 0.1 0.21 0.24 0.25 Ileum 3.3 ± 0.33.6 ± 0.1 3.6 ± 0.1 3.7 ± 0.1 0.35 0.36 0.31 Cecum 2.8 ± 0.2 3.5 ± 0.13.6 ± 0.1 3.4 ± 0.1 0.11 0.05 0.10 Colon 3.6 ± 0.3 3.5 ± 0.1 3.3 ± 0.13.5 ± 0.1 0.71 0.46 0.76 Rectum 3.0 ± 0.7 3.0 ± 0.2 3.0 ± 0.2 3.4 ± 0.20.98 0.98 0.64 ^(A) 0: severely abnormal; 1: markedly abnormal, 2:moderately abnormal, 3: mildly abnormal; 4: normal (see SupplementaryMethods). Sample-average scores were calculated for each sample overevaluated histological sub-structures (villi/epithelium, crypts, laminapropria/submucosa, Brunner's glands). Mean sample-average scores pergroup are shown. ^(B) Student's t-test vs. vehicle, 2-tailed.

To assess the effect of entolimod on the GI component of ARS during thepeak of GI damage, three additional dedicated NHP studies were designed(Table 10). In these studies, irradiated animals (a total of 48 NHPs,equal numbers of males and females) were euthanized at different timepoints between 8 hours and 7 days after TBI, when signs of immediate andearly radiation-induced GI damage are typically observed along withindications of the initiation of recovery processes. The animalsreceived TBI doses sufficient to induce moderate to severe GI injury(6.5-11 Gy, expected to cause 70-100% mortality) and entolimod dosesranging between 0.3 and 40 μg/kg at 1 to 25 hours after TBI. Someanimals received EdU injections prior to euthanasia to allow evaluationof crypt proliferation by visualization of EdU incorporation onhistological sections.

TABLE 10 Layout of studies dedicated to assessment of RS entolimodeffects on GI tract histopathology in the course of ARS IrradiationInjection Timing of dose Entolimod time scheduled Study and dose,relative Group euthanasia Number source μg/kg to TBI, h size (after TBI)Rs-04 ~LD_(75/40) 0 (vehicle) +1 2 8 h (6.5 Gy); 40 +1 2 8 h Co60^(A) 0(vehicle) +1 2 5 d 40 +1 2 5 d Rs-08 ~LD_(75/40) 0 (vehicle) +1 2 8 h(6.5 Gy); 3 +1 2 8 h Co60^(A) 10 +1 2 8 h 40 +1 4 8 h 100 +1 2 8 h 200+1 2 8 h 0 (vehicle) +1 2 5 d 3 +1 2 5 d 10 +1 2 5 d 40 +1 4 5 d 100 +12 5 d 200 +1 2 5 d 40 +16 2 5 d 40 +25 2 5 d Rs-22 >LD_(95/40) 0(vehicle) +4 4 7 d^(C) (11 Gy); 40 +4 4 7 d^(C) LINAC^(B) ^(A)Source I:Sichuan Atomic Energy Institute, cylindrical bundle of cobalt rods^(B)Source III: UI CTRL, 6 MV LINAC source (Varian Clinac 2100EX) ^(C)10mg/kg EdU, i.v. 1 h before euthanasia

At 8 hours after 6.5 Gy TBI, the number of apoptotic cells counted in˜200 small intestine crypts was ˜4.2-fold lower in animals treated withentolimod 1 hour after TBI (˜1.16 TUNEL-positive cells/crypt) comparedto vehicle-treated animals (˜4.74 TUNEL-positive cells/crypt) (FIG. 9,panel A). There were only a few apoptotic cells in the crypts of thelarge intestine and rectum (˜0.3 cells/crypt) regardless of treatment.Administration of entolimod also resulted in more robust expression ofthe NF-κB-regulated anti-oxidant enzyme SOD2 in small intestine villi,crypts and lamina propria (FIG. 9, panel B). Entolimod-treated (1 hourafter TBI) NHPs showed improved morphology in all analyzed GI segmentsat 5 days after exposure to 6.5 Gy TBI compared to vehicle-treatedcontrols (FIG. 10). In addition to showing improvedpreservation/recovery of intestinal villi, crypts, and lymphoidaccumulations in the lamina propria, entolimod-treated animalsdemonstrated better preservation of elements of the intestinal nervoussystem and muscularis mucosa (FIG. 11). The mitigative effect ofentolimod on radiation-induced injury to the GI tract was inverselyproportional to the time interval between irradiation and drugadministration, but was still clearly observed even when the drug wasgiven 25 hours after TBI (FIG. 10). At 7 days after 11 Gy TBI,irradiated and entolimod-treated (40 μg/kg, at 4 hours after TBI) NHPs,unlike vehicle-treated animals, demonstrated massive crypt regenerationas indicated by robust EdU incorporation (FIG. 9, panel C) and tissuemorphology (microcolony growth visible by light microscopy, FIG. 9,panel D). Parallel semiquantitative blind histological assessment ofradiation injury in different tissue elements throughout the small andlarge intestines showed statistically significant differences,indicating a beneficial effect of entolimod treatment (Table 11 andTable 12).

TABLE 11 Histological evaluation of GI tract segments on day 7 after 11Gy TBI and vehicle or 40 μg/kg entolimod treatment at +4 hours (studyRs-22, N = 4/group) Vehicle score ^(A), Entolimod score ^(A), T-testP-value GI segment mean ± SE mean ± SE (E vs. V) ^(B) Oral Mucosa 0.9 ±0.03 1.5 ± 0.06 0.001 Esophagus 1.1 ± 0.03 1.7 ± 0.09 0.01 Stomach 1.1 ±0.05 2.3 ± 0.08 0.0001 Duodenum 1.0 ± 0.05 1.6 ± 0.03 0.0004 Jejunum 0.9± 0.07 1.3 ± 0.12 0.05 Ileum 1.1 ± 0.07 1.3 ± 0.02 0.04 Cecum 1.1 ± 0.071.3 ± 0.03 0.08 Ascending Colon 0.9 ± 0.17 1.5 ± 0.15 0.05 TransverseColon 1.0 ± 0.17 1.4 ± 0.02 0.08 Descending Colon 0.9 ± 0.19 1.3 ± 0.030.12 Rectum 1.1 ± 0.03 1.2 ± 0.05 0.15 ^(A) 0: severely abnormal; 1:markedly abnormal; 2: moderately abnormal; 3: mildly abnormal; 4: normal(see Supporting Information, SI Methods). ^(B) Student's t-test ofentolimod (E) vs. vehicle (V) scores, 2-tailed

TABLE 12 Semi-quantitative histological evaluation of GI tract sub-structures on day 7 after 11 Gy TBI and vehicle or 40 ug/kg entolimodtreatment at 4 h post-TBI (study Rs-22, N = 4) 40 μg/kg IntestinalVehicle score ^(A), Entolimod score ^(A), T-test P-value structure mean± SE mean ± SE (E vs. V) ^(B) MALT^(C) 1.0 ± 0.03 1.4 ± 0.04 <0.0001Lamina Propria 1.0 ± 0.04 1.3 ± 0.03 <0.0001 Surface 1.1 ± 0.06 1.4 ±0.03 <0.0001 Epithelium Crypts 1.1 ± 0.07 1.4 ± 0.06 <0.0001 Villi 0.9 ±0.09 1.3 ± 0.06 <0.0001 ^(A) 0: severely abnormal; 1: markedly abnormal;2: moderately abnormal; 3: mildly abnormal; 4: normal (see SupplementaryMethods). ^(B) Student's t-test of 40 μg/kg entolimod (E) vs. vehicle(V) scores, 2-tailed. ^(C)Mucosa-associated lymphoid tissue.

Without wishing to be bound by theory, it is believed that entolimodeffectively mitigates radiation injury to the GI system, likely actingvia reduction of apoptosis and stimulation of regeneration.

Pharmacokinetics and Pharmacodynamic Effects of Entolimod in LethallyIrradiated NHPs

In the NHP studies reported here, plasma levels of numerous cytokineswere measured at multiple time points following entolimod treatment.G-CSF and IL-6, previously established as potential entolimod efficacybiomarkers, displayed the most substantial and consistent dose-dependentresponses to the drug when administered after LD_(50-75/40) TBI doses,with levels peaking on average at 2-4 hours after drug administration(FIG. 12). These results are similar to observations made innon-irradiated NHPs and NHPs irradiated with LD_(20-30/40) TBI doses.Both of these cytokines were induced somewhat by radiation alone (FIGS.12, A and B); therefore, treatment with entolimod close to TBI (e.g., at1 hour after TBI) resulted in a combined effect of both treatments onthe magnitude of cytokine increase. When entolimod was administered at25 hours after TBI (after dissipation of radiation-induced G-CSF andIL-6 responses in vehicle-treated animals), entolimod-elicited cytokineprofiles were more similar to those seen in non-irradiated animals (FIG.12, C-F).

Among other cytokines previously shown to be substantially influenced byentolimod in non-irradiated or sublethally irradiated (LD_(20-30/40)TBI) NHPs, IL-8, with neutrophil-mobilizing activity, and IL-10, withanti-inflammatory activity, are also worth mentioning (FIG. 13). Bothfactors were found to be strongly responsive to entolimod and, to someextent, also to TBI. However, the magnitude of their elevation byentolimod and its dependence on drug dose were less consistent amongdifferent studies compared to G-CSF or IL-6. Neither radiation norentolimod elicited any apparent response in pro-inflammatory cytokinessuch as IL-2, IP-10, IL-12p70, IL-4, IFNγ, or IL-3.

The pharmacokinetics of entolimod was similar in irradiated andnon-irradiated NHPs, with C_(max) and AUC₀₋₂₄ values being very close atidentical drug doses. In both irradiated and non-irradiated NHPs,measured concentrations and exposures of entolimod in the blooddisplayed clear dose dependence (FIG. 14).

Necropsy Findings

Gross pathology findings at necropsy of animals that succumbed to ARSbefore Day 40 were generally consistent with those expected from ARSpathogenesis and consisted of hemorrhages (of varying extents anddegrees of severity, mainly observed in the skin, GI tract, lungs andpericardium), septic complications (mainly in the lungs, pericardium,and skin), and generalized sepsis with multiple organ involvement. Amongfrequent findings, there were intussusceptions in the small and largeintestines, adhesions in the abdominal and thoracic cavities, and signsof lung edema. There were no marked differences in gross pathologyfindings between entolimod- and vehicle-treated animals. Thisobservation is not unexpected since entolimod treatment did not succeedin mitigating radiation damage in the animals that were necropsiedduring the course of the study, as evidenced by their mortality. At thesame time, NHPs in which entolimod was actually effective (leading totheir survival until study termination on Day 40 post-TBI—40-60% ofanimals at doses 10 μg/kg) could not be assessed for gross pathologystatus at the time of early recovery from ARS injury.

Gross pathology findings in animals that survived to the end of thestudy and were euthanized on Day 40-41 post-TBI were minimal regardlessof treatment group, indicating that post-ARS recovery was generallycomplete by that time point. This observation was consistent with thelack of mortality from ARS after day 30 in all study groups.

The results described here demonstrate that entolimod possesses all ofthe aforementioned desirable properties for an MRC. In fact, when givento NHPs as a single agent (without additional intensive supportive care)via a simple i.m. injection up to 48 hours after TBI, entolimod had astrong and consistent radiomitigative effect in four independentexperiments involving 164 NHPs. Overall, entolimod treatment reduced therisk of NHP death 2-3-fold at TBI doses of LD_(50-75/40), providing anabsolute survival advantage of 40-60% over vehicle treatment. In twoadditional studies with a total of 82 NHPs exposed toLD_(20/40)-LD_(30/40) TBI doses, single injection of entolimod within atleast 48 hours after TBI increased survival by 20-31%: from 69-80% invehicle-treated control groups to 100% in entolimod-treated groups.Although the magnitude of achievable survival improvement in theselatter two studies was limited by low lethality of the TBI doses used,the survival odds ratios were 4.75. The NHP studies reported hereclearly show that entolimod treatment in the context of lethal TBI leadsto reduced damage and accelerated recovery in both the radiosensitive HPand GI systems.

Binding of entolimod to TLR5 results in stimulation of a number ofdownstream pathways, including those regulated by the key TLR5-activatedtranscription factor, NF-κB. Ultimately, as shown in the non-limitingmodel in FIG. 15, this engages multiple mechanisms of action against themulti-faceted toxic effects of ionizing radiation.

Without wishing to be bound by theory, one of these mechanisms appearsto be neutralization of radiation-triggered reactive oxygen species(ROS) by the enzyme superoxide dismutase (SOD2), which is stronglyinduced by entolimod in both mouse and NHP models of ARS. Anothermechanism that is a likely contributor to entolimod's radioprotectiveand radiomitigative effects, without wishing to be bound by theory, isinhibition of radiation-induced apoptosis, which is well-recognized as amajor cause of the tissue damage and cytopenias observed in ARS.Anti-apoptotic effects of entolimod are likely mediated via induction ofNF-κB and its downstream anti-apoptotic effectors, such as members ofthe IAP and Bcl-2 protein families. Additional anti-apoptotic mechanismsof entolimod may include direct activation of the PI3K pathway and of aspecific anti-apoptotic phosphatase, MKP7, recently identified as aninhibitor of radiation-dependent GI cell apoptosis, both triggered byflagellin stimulation of TLR5. Entolimod was shown to reduceradiation-induced apoptosis of cells in GI tissues both in mice andNHPs. It may also attenuate apoptosis of other types of cells relevantto development of ARS such as inhibition of neutrophil apoptosis. Inaddition, stimulation of TLR5 is expected to inhibit radiation-inducedaseptic inflammation involved in secondary apoptotic tissue damage e.g.via induction of an anti-inflammatory cytokine IL-10, IL-1β antagonist(IL-1βa) and stimulation of mesenchymal stem cells (MSC) known toexpress TLR5 and to have anti-inflammatory properties.

Restoration of the integrity and functionality of damaged organsfollowing irradiation depends on the availability of sufficient numbersof undamaged tissue stem cells. The ability to protect and stimulatestem cells is expected to be an important property of any effectiveradiomitigator. This study demonstrated protective and stimulatoryeffects of entolimod on stem cells in both HP and GI tissues.Entolimod-treated irradiated NHPs displayed increased clonogenicpotential of the BM and improved survival of intestinal-crypt stem cellsas indicated by robust and accelerated crypt proliferation. Thebeneficial effects of entolimod on HP and GI stem cells are translatedinto facilitation of morphological recovery of the correspondingtissues. Without wishing to be bound by theory, it is believed that themechanism(s) underlying entolimod's stimulatory effects on stem cellsare likely mediated by induced cytokines, some of which are known topossess this activity. Among the cytokines elevated in response toentolimod, two hematopoietic cytokines, G-CSF and IL-6, consistentlyshowed the strongest induction. The importance of these cytokines forthe radiomitigative activity of entolimod has been proven experimentallyin vivo using neutralizing antibodies and is fully consistent with theirdefined biological activities as stimulators of granulo- andthrombopoiesis, respectively. Consequently, both the severity andduration of radiation-induced thrombocytopenia and neutropenia weresignificantly reduced in entolimod-treated NHPs. Entolimod's promotionof red blood cell lineage recovery (reticulocytes) with kinetics andmagnitude similar to its effects on thrombocytopenia may be suggestiveof stimulation of megakaryocyte/erythrocyte-restricted progenitors(MEPs). The combined effects of entolimod on reducing the severity andduration of thrombocytopenia, and accelerating recovery of the erythroidlineage result in markedly diminished incidence of life-threateningGrade 4 hemorrhagic anemia, one of the hallmarks of HP ARS.

Loss of tissue integrity due to TBI leads to development of wounds andseptic complications, which are especially dangerous on the backgroundof impaired tissue repair and immunosuppression. Anti-infectiveproperties reported for flagellin (and likely also relevant forentolimod due to its similar mechanism of action) are consistent withits general role as a trigger of TLR5-mediated innate immune response tobacterial infection. Indeed, flagellin was shown to induce secretion ofantimicrobial factors, such as IL-17, S100A8/S100A9, hepcidin and othersmall peptides with antimicrobial activity, to support anti-infectivedefenses and tissue repair in the lungs, gut, skin and cornea. Directanti-bacterial activity of flagellin (or a flagellin variant with astructure similar to that of entolimod) has been demonstrated in animalinfection models. This activity was likely associated with the abilityof flagellin/entolimod to elicit early neutrophil mobilization (observedeven in irradiated NHPs within 24 hours following entolimodtreatment—see FIGS. 4, C and D; and FIGS. 5, C and D) followed byneutrophil infiltration into tissues where they play an important rolein local antibacterial responses. Mobilization and tissue deposition ofneutrophils (especially in the lung and the liver) can be explained byboth entolimod-dependent induction of IL-8 and by entolimod's directaction on TLR5 expressed on the surface of neutrophils. TLR5 activationalso enhances the phagocytic capacity and the respiratory burst activityof airway neutrophils, which likely contributes to their antibacterialpotency. At later times in the course of ARS, entolimod-inducedaccelerated recovery from radiation cytopenias would also be expected tocontribute to antibacterial immunity (via restoredgranulocyte/macrophage function) and wound healing (via restored bloodclotting and tissue trophic function of platelets). Another mechanismthrough which entolimod may promote wound healing, without wishing to bebound by theory, is direct stimulation of fibroblasts and MSC.

Example 3: Dosing of CBLB502 is Safe in Humans

A clinical trial to assess that, in healthy human subjects, the proposeddosing regimen of CBLB502 is safe is undertaken. CBLB502 or placebo isadministered to human subjects on a weight-adjusted basis withinspecific ranges of body weights to 1) describe the safety profile of thedrug using this regimen, and 2) assess the PD effects of the drug onrelevant biomarkers (in particular G-CSF, IL-6, and fold-change in ANC)using this regimen.

Trial subjects are healthy adult male and non-pregnant female subjects18 years. Healthy subjects are considered a relevant population becausethe majority of the victims of a radiological event will have beenhealthy individuals prior to IR exposure. Subjects are enrolled instrata based on body weight and randomized in a 6:1 ratio to receive asingle IM injection of CBLB502 or placebo according to the followingTable 13:

TABLE 13 Dose Intervals Designed to Ensure Body-Weight-Adjusted Dosingwithin Range of 0.40-0.60 μg/kg Body-Weight- Vial Size, Adjusted μgDose, μg/kg 35 Body Weight, Body Weight, At At Vial kg lb Mini- Maxi-Amount, Absolute Mini- Maxi- Mini- Maxi- mum mum μL Dose, μg mum mum mummum Weight Weight 20 2 4 5 9 11 0.50 0.40 30 3 6 8 13 17 0.50 0.40 50 59 13 19 28 0.59 0.40 80 8 14 20 30 44 0.59 0.40 120 12 21 30 46 66 0.570.40 130 13 31 33 68 72 0.42 0.40 200 20 34 50 74 110 0.60 0.40 300 3051 75 112 165 0.59 0.40 450 45 76 113 167 248 0.59 0.40 450 45 >114∞ >250 ∞ 0.40 <0.40

The study is performed at a specialized inpatient clinic located in theUS that is experienced in the conduct of trials in healthy subjects.Subjects undergo screening medical history, physical examination, vitalsigns, laboratory evaluation, and electrocardiogram (ECG) assessments.Subjects are administered CBLB502 or placebo IM in the morning of Day 1and then observed as inpatients for 36 hours. Types of adverse eventsare coded using the standard Medical Dictionary for RegulatoryActivities (MedDRA) and severity is graded using the Common TerminologyCriteria for Adverse Events (CTCAE), Version 4.03. Adverse eventfrequency, timing of onset duration and relationship to study therapy isalso recorded. Laboratory abnormalities are recorded. To describehemodynamic changes that are expected with administration of a TLRagonist, supine and sitting blood pressures is assessed predose, hourlyfor 12 hours postdose, and then every 4 hours through 36 hours postdose.ECGs, clinical chemistry and hematology data, and plasma forcytokines—including G-CSF, IL-6, as well as ILs-1α, -1β, -2, -8, -10,-12, tumor necrosis factor-α, interferon (IFN)-β and IFN-γ, is obtainedpredose and at 1, 2, 4, 8, 12, 24 hours postdose to fully describe theCBLB502 safety, PD, and inflammatory profiles. Serum nitrate levelsobtained at the same time points may offer correlates to changes inblood pressure. Subjects undergo follow-up at Days 8 and 29 forassessment of vital signs, ECGs, safety, PD parameters, and anti-CBLB502antibodies. The sample size for the study provides a large statisticalsample (i.e., 30 subjects) for each dosing regimen. Subjectcharacteristics and study results are described by dosing group usingtabular and graphical methods to support CBLB502 product labeling.

Example 4: CBLB502 can Reduce the Risk of Death Following not Only LD70,but Also LD30 and LD50 Doses of Total Body Irradiation (TBI)

Studies are undertaken to provide statistically robust data on theefficacy of CBLB502 in non-human primates (NHPs) over an LD30-50 rangeof TBI doses. These radiation doses will be used to complement priorpivotal efficacy study performed at LD70/60 and provide efficacy testingat LD30, LD50, and LD70 TBI doses. Four groups of NHPs (˜1:1 male:femaleratio) receive a single dose of TBI: LD30/60 (Groups 1, 2: 36animals/group) or LD50/60 (Groups 3, 4: 30 animals/group), followed 25hours later with a single IM injection of vehicle (Groups 1, 3) or theanimal doses of CBLB502 of 10 μg/kg (Groups 2, 4). The study israndomized, blinded, and placebo-controlled. 60-day survival is theprimary endpoint. The sample sizes (N=36 for LD30, N=30 for LD50 TBIlevels) provide power >0.95 (α=0.05, 2-sided, Cochran-Mantel-Haenszeltest) to detect an CBLB502 60-day survival benefit. These sizes alsoprovide a power of 0.80 for descriptive comparisons of the proportionsof survivors at each TBI level (α=0.05, 2-sided, Fisher's exact test).PK and PD effects on biomarkers and blood counts are evaluatedsecondarily. This study also looks for correlations between biomarkerand hematopoietic responses to drug and the time dependence of efficacy.This study is performed under GLP with elements of GCP, includingblinding, randomization, and pre-specified statistical plan. All PD andPK measurements are obtained using GLP-validated assays.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

1.-32. (canceled)
 33. A method of preventing gastrointestinal damagefollowing exposure to radiation in a human patient, the methodcomprising administering to the human patient not more than a singledose of an effective amount of a composition comprising entolimod,wherein the effective amount is about 0.4 to about 0.6 μg/kg, andwherein the entolimod comprises the amino acid sequence of SEQ ID NO: 1.34. The method of claim 33, wherein the exposure to radiation occurs asa result of a radiation disaster.
 35. The method of claim 33, whereinthe irradiation is a high dose of radiation of at least 2 Gy.
 36. Themethod of claim 33, wherein the high dose of radiation is sufficient fora classification of Unit Radiation Exposure Status of RES
 3. 37. Themethod of claim 33, wherein the human patient is administered entolimodwithin about 48 hours of being exposed to radiation.
 38. The method ofclaim 33, wherein the human patient is administered entolimod within oneor more of the triage, emergency care, and definitive care stages ofradiation exposure.
 39. The method of claim 33, wherein entolimod isadministered parenterally or orally.
 40. The method of claim 33, whereinentolimod is administered by a single intramuscular injection.
 41. Themethod of claim 33, wherein entolimod is administered bycontrolled-release or sustained-release.
 42. The method of claim 33,wherein the human patient presents a lymphocyte count reduction of about50% within about 24 to about 48 hours of being exposed to radiation. 43.The method of claim 33, wherein the human patient's lymphocyte count isless than about 1000/μL.
 44. The method of claim 33, wherein thetreatment with entolimod is used as an adjuvant or neoadjuvant to one ormore of blood products, colony stimulating factors, cytokines and/orgrowth factors, antibiotics, diluting and/or blocking agents, mobilizingor chelating agents, stem cell transplants, antioxidants or freeradicals, and radioprotectants.
 45. The method of claim 33, whereinentolimod induces expression of NF-κB through toll-like receptor(TLR)-mediated activation.